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A major barrier to adeno-associated virus (AAV) gene therapy is the inability to re-dose patients due to formation of vector-induced neutralizing antibodies (Nabs). Tolerogenic nanoparticles encapsulating rapamycin (ImmTOR) provide long-term and specific suppression of adaptive immune responses, allowing for vector re-dosing. Moreover, co-administration of hepatotropic AAV vectors and ImmTOR leads to an increase of transgene expression even after the first dose. ImmTOR and AAV Anc80 encoding the methylmalonyl-coenzyme A (CoA) mutase (MMUT) combination was tested in a mouse model of methylmalonic acidemia, a disease caused by mutations in the MMUT gene. Repeated co-administration of Anc80 and ImmTOR was well tolerated and led to nearly complete inhibition of immunoglobulin (Ig)G antibodies to the Anc80 capsid. A more profound decrease of plasma levels of the key toxic metabolite, plasma methylmalonic acid (pMMA), and disease biomarker, fibroblast growth factor 21 (FGF21), was observed after treatment with the ImmTOR and Anc80-MMUT combination. In addition, there were higher numbers of viral genomes per cell (vg/cell) and increased transgene expression when ImmTOR was co-administered with Anc80-MMUT. These effects were dose-dependent, with the higher doses of ImmTOR providing higher vg/cell and mRNA levels, and an improved biomarker response. Combining of ImmTOR and AAV can not only block the IgG response against capsid, but it also appears to potentiate transduction and enhance therapeutic transgene expression in the mouse model., Graphical abstract, Kishimoto and colleagues extend their earlier findings showing that combining ImmTOR rapamycin-encapsulated nanoparticles with hepatotropic AAV vector leads to increased transduction, transgene expression, and suppression of IgG responses and therefore enables effective therapeutic AAV vector re-dosing in a mouse model of methylmalonic acidemia, an inborn metabolic disease.

DNA testing of human bones is performed for identification when there is no remaining soft tissue, which often means the samples are old or environmentally compromised. Under these circumstances, it can be difficult to obtain a STR DNA profile. It is important to recover the highest quantity and quality of DNA for STR typing. This study compared the DNA recovery and STR profiles from five anatomical locations in five femora and five tibiae. These locations include the proximal metaphysis, proximal diaphysis, mid-diaphysis, distal diaphysis, and distal metaphysis. Twenty-five femur samples and 25 tibia samples were analyzed using the Qiagen Investigator Quantiplex Pro RGQ Kit for quantitating the extracted DNA and the Qiagen Investigator 24plex QS Kit for STR DNA typing. The highest DNA recovery of the five regions tested in both the femur and the tibia was from the midshaft diaphysis. The femur samples resulted in a significantly higher DNA recovery than the tibia samples as analyzed using a Kruskal-Wallis test (P = 0.002103). The midshaft diaphysis and distal diaphysis yielded the most complete STR DNA profiles in the femora, while the distal and proximal diaphysis yielded the most complete STR DNA profiles in the tibiae. There was no correlation between the amount of DNA recovered and the completeness of the STR DNA profile produced with low template extracts in this study.

Supplemental light can increase growth and accelerate production of greenhouse crops, but it can be expensive if not provided in a way that promotes efficient use of the light. Dimmable light-emitting diode (LED) fixtures have the potential to reduce lighting costs because the output can be precisely controlled to meet crop needs. Because light is used more efficiently to drive photosynthesis at lower photosynthetic photon flux densities (PPFDs), we hypothesized that providing Rudbeckia fulgida var. sullivantii ‘Goldsturm’ seedlings with the same daily light integral (DLI), spread out over a longer photoperiod and at lower PPFDs, should improve growth. A DLI of 12 mol·m−2·d−1 was provided in a greenhouse over 12, 15, 18, or 21-hour photoperiods from a combination of sunlight and supplemental light from LEDs, using adaptive lighting control. Plants grown without supplemental light had an ≈12-hour photoperiod and received an average DLI of 5 mol·m−2·d−1, ≈58% less light than the four lighting treatments. Lengthening the photoperiod from 12 to 21 hours increased shoot dry mass (30%), root dry mass (24%), plant height (14%), leaf area (16%), and chlorophyll content index (48%), and decreased specific leaf area (26%). There was no significant effect of photoperiod on root mass fraction or compactness. Growth parameters of plants without supplemental light were 26% to 90% smaller compared with those in the 12-hour photoperiod treatment. Treatment effects on canopy size, seen as early as 2 weeks into the study, were correlated with final shoot dry mass. Longer photoperiods did not induce a shade-avoidance response, based on specific leaf area and compactness data. The 24% increase in root dry mass for the plants in the 21-hour photoperiod suggests that cropping cycles can be shortened by 1 to 2 weeks compared with the 12-hour photoperiod. This could result in more crop turns per year and increased profits. In addition, fewer lights would be needed for adequate growth, reducing the capital cost of the lighting system.

A quick, simple, and high-yield nucleic acid isolation process is crucial for high-quality DNA analysis. The ability of the MicroGEM PDQeX phytoGEM system and Omega Bio-tek E.Z.N.A.® Plant DS Mini kit to extract PCR-ready DNA was evaluated by extracting the forensically relevant "legal high" plant species: Ipomoea purpurea, Artemisia absinthium, Mitragyna speciosa, Datura stramonium, and Papaver somniferum. The plant material was pulverized, processed using the manufacturer's plant protocol for the PDQeX Nucleic Acid Extraction or the manufacturer's protocol for the Omega extraction, quantified using the Invitrogen Qubit 2.0 Fluorometer, and analyzed for amplifiability by PCR using a Qiagen Rotor-Gene Q instrument and published assays. The DNA amplicons for the legal high species produced high-resolution melt curves concordant with the melts observed when DNA was isolated using the Qiagen DNeasy Plant Mini Kit in previous studies.

Carbapenemase gene-positive (CP) Gram-negative bacilli are of significant clinical and public health concern. Their rapid detection and containment are critical to preventing their spread and additional infections they can cause. To this end, CDC developed the Antibiotic Resistance Laboratory Network (AR Lab Network), in which public health laboratories across all 50 states, several cities, and Puerto Rico characterize clinical isolates of carbapenem-resistant Enterobacterales (CRE), Pseudomonas aeruginosa (CRPA), and Acinetobacter baumannii (CRAB) and conduct colonization screens to detect the presence of mobile carbapenemase genes. In its first 3 years, the AR Lab Network tested 76,887 isolates and 31,001 rectal swab colonization screens. Targeted carbapenemase genes (bla(KPC), bla(NDM), bla(OXA-48-like), bla(VIM), or bla(IMP)) were detected by PCR in 35% of CRE, 2% of CRPA, and

The polymerase chain reaction (PCR) is used to copy DNA in vitro for a variety of applications including amplifying a target DNA, mutating a base, adding tags, and sequencing by synthesis applications. Next-generation sequencing (NGS) is a DNA sequencing technology that has been applied to screening cancer and tissue variants, deep sequencing, and gene expression analysis, and more recently, it has been applied to DNA typing for human identification, estimating age, and detecting and differentiating body fluids. Body fluids are normally identified using color tests, microscopy, and immunochromatographic assays. Pyrosequencing is an NGS approach that has been applied to body fluid analysis. The pyrosequencing assays can detect one or several mixed body fluids by analysis of their tissue-specific differentially methylated regions (tDMRs). Here, the process of designing pyrosequencing primers for forensic biology applications is described.

The polymerase chain reaction (PCR) is used to copy DNA in vitro for a variety of applications including amplifying a target DNA, mutating a base, adding tags, and sequencing by synthesis applications. Next-generation sequencing (NGS) is a DNA sequencing technology that has been applied to screening cancer and tissue variants, deep sequencing, and gene expression analysis, and more recently, it has been applied to DNA typing for human identification, estimating age, and detecting and differentiating body fluids. Body fluids are normally identified using color tests, microscopy, and immunochromatographic assays. Pyrosequencing is an NGS approach that has been applied to body fluid analysis. The pyrosequencing assays can detect one or several mixed body fluids by analysis of their tissue-specific differentially methylated regions (tDMRs). Here, the process of designing pyrosequencing primers for forensic biology applications is described.

Fire blight-susceptible, certified organic pome fruit is produced on 9,000 ha in the Pacific Northwest region of the United States with acreage continuing to expand despite a 2014 prohibition on antibiotics as allowable materials for infection suppression. Nonantibiotic practices for fire blight pathogen suppression mirror conventional management, but the full-bloom-to-petal-fall period when antibiotics are typically sprayed for fire blight control continues to receive research scrutiny owing to drawbacks and weaknesses of alternative materials. As solitary treatments, effective nonantibiotic materials (e.g., a yeast biocontrol, soluble coppers, and potassium aluminum sulfate) raise the risk of a crop-value–reducing, phytotoxic response termed “fruit russeting.” Conversely, materials with less russeting risk (e.g., Bacillus-based biorationals) are less effective for fire blight control. Spray programs using a sequence of materials applied from midbloom to petal fall have the potential to provide high levels of protection with reduced russeting risk. In orchard trials, the effects of nonantibiotic spray programs on the epiphytic population size of Erwinia amylovora in flowers, yeast biocontrol population size, floral pH, infection suppression, and fruit russeting revealed strategies for sequencing sprays of nonantibiotic materials. The yeast biocontrol, Blossom Protect (Aureobasidium pullulans), sprayed at 70% bloom, was an important contributor to fire blight pathogen suppression as was the soluble copper material, Previsto, when applied at full bloom. Choice of material for the petal-fall spray timing was important to fruit russeting risk but apparently less important to overall infection incidence. Consequently, treatment programs of Blossom Protect at 70% bloom, a soluble copper at full bloom, and a Bacillus-based biorational at petal fall, best balance the quality of infection suppression with the risk of fruit russeting.

Vulcanodinium is an ecologically relevant dinoflagellate genus due to its production of neurotoxins known as pinnatoxins. We present here the first examination of the sterols of a Vulcanodinium rugosum isolate. Sterols are ringed lipids that assist in maintaining rigidity of cellular membranes, and the Dinophyceae are well-studied for their ability to produce a diverse array of sterols, many of which have chemotaxonomic utility. We have determined that V. rugosum produces a set of major sterols, namely cholesterol, dinosterol, 4α,24-dimethyl-5α-cholest-22E-en-3β-ol, and 4α,24-dimethyl-5α-cholestan-3β-ol, common to the Dinophyceae. However, this displayed marked differences from those studied members of the genera Scrippsiella and Peridinium, the closest phylogenetic relatives. Included in these differences is production by V. rugosum of a much lower percentage of dinostanol, a saturated form of dinosterol.

Sorghum (Sorghum bicolor (L.) Moench) is an important summer grain crop in the U.S. Southern Great Plains because it is one of the few crops that consistently produces acceptable yields in the harsh summer weather that characterizes the region. Damaging infestations of the sugarcane aphid, Melanaphis sacchari (Zehntner), occur commonly in sorghum throughout Texas and Oklahoma, two of the leading sorghum producing states. Grain sorghum fields were sampled at two locations in Texas and three locations in Oklahoma. Fields were sampled approximately weekly by examining two leaves on each of 54 plants and counting all sugarcane aphids and aphid natural enemies on each leaf. Sampling began at an early growth stage and continued until the crop either matured or was treated with insecticide to suppress the sugarcane aphid infestation. For a total of 123 fields in 2017 and 2018, aphids, Aphelinus nigritus Howard mummies, adult coccinellids, larval coccinellids, larval lacewings, and larval dipterans were counted, and relative density estimates were determined. When natural enemy and sugarcane aphid count data were aggregated at the scale of geographic locations and multiple years, there was no evidence for a numerical response by natural enemies to sugarcane aphid density. When fields were compared within locations, a numerical response was consistently observed. A natural enemy importance index was developed that incorporated cumulative degree days of first occurrence of a natural enemy taxa in a field, the average density of the natural enemy in a field, and natural enemy voracity. Factorial analyses of variance indicated that cumulative degree days at first occurrence and average relative density differed significantly among natural enemy taxa and locations, as did natural enemy importance. Averaged across locations larval coccinellids had the largest importance index, I = 1.47, and A. nigritus had the smallest, I = 0.61. Among locations, the Texas Coastal Plains had the largest importance index, I = 1.27 whereas the Oklahoma Panhandle had the smallest, I = 1.02. Results suggest that differences occur in the biological control contributions of various natural enemies and in biological control efficacy among locations.

Inhalation of small numbers of Francisella tularensis subspecies tularensis (Ftt) in the form of small particle aerosols causes severe morbidity and mortality in people and many animal species. For this reason, Ftt was developed into a bona fide biological weapon by the USA, by the former USSR, and their respective allies during the previous century. Although such weapons were never deployed, the 9/11 attack quickly followed by the Amerithrax attack led the U.S. government to seek novel countermeasures against a select group of pathogens, including Ftt. Between 2005–2009, we pursued a novel live vaccine against Ftt by deleting putative virulence genes from a fully virulent strain of the pathogen, SCHU S4. These mutants were screened in a mouse model, in which the vaccine candidates were first administered intradermally (ID) to determine their degree of attenuation. Subsequently, mice that survived a high dose ID inoculation were challenged by aerosol or intranasally (IN) with virulent strains of Ftt. We used the current unlicensed live vaccine strain (LVS), first discovered over 70 years ago, as a comparator in the same model. After screening 60 mutants, we found only one, SCHU S4 ΔclpB, that outperformed LVS in the mouse ID vaccination-respiratory-challenge model. Currently, SCHU S4 ΔclpB has been manufactured under current good manufacturing practice conditions, and tested for safety and efficacy in mice, rats, and macaques. The steps necessary for advancing SCHU S4 ΔclpB to this late stage of development are detailed herein. These include developing a body of data supporting the attenuation of SCHU S4 ΔclpB to a degree sufficient for removal from the U.S. Select Agent list and for human use, optimizing SCHU S4 ΔclpB vaccine production, scale up, and long-term storage, and developing appropriate quality control testing approaches.

Artemisia absinthium (wormwood), a common ingredient in absinthe, contains the compound thujone, which is unregulated by the U.S. Drug Enforcement Agency. Thujone can cause an “unregulated legal high” in higher concentrations. The European Union limits thujone from Artemisia species to 35 mg/kg while the U.S. Food and Drug Administration requires less than 10 ppm to be “thujone-free.” However, individuals can smoke or ingest A. absinthium in different forms. This study developed a polymerase chain reaction (PCR) highresolution melt (HRM) assay to detect and identify A. absinthium based on primer specificity, sensitivity, repeatability, and robustness. A triplex assay was performed with three “unregulated legal high” species: Datura stramonium, Merremia tuberosa, and A. absinthium; the PCR HRM assay detected and identified each plant at melt temperatures 77.42+-0.20°C, 83.88+-0.22°C, and 87.77+- 0.15°C, respectively. The primer set developed distinguished A. absinthium from a variety of plant species and was successfully triplexed.

Disrupted serotonergic neurotransmission has long been implicated in major depressive disorder (MDD), for which selective serotonin reuptake inhibitors (SSRIs) are the first line of treatment. However, a significant percentage of patients remain SSRI-resistant and it is unclear whether and how alterations in serotonergic neurons contribute to SSRI resistance in these patients. Induced pluripotent stem cells (iPSCs) facilitate the study of patient-specific neural subtypes that are typically inaccessible in living patients, enabling the discovery of disease-related phenotypes. In our study of a well-characterized cohort of over 800 MDD patients, we generated iPSCs and serotonergic neurons from three extreme SSRI-remitters (R) and SSRI-nonremitters (NR). We studied serotonin (5-HT) biochemistry and observed no significant differences in 5-HT release and reuptake or in genes related to 5-HT biochemistry. NR patient-derived serotonergic neurons exhibited altered neurite growth and morphology downstream of lowered expression of key Protocadherin alpha genes as compared to healthy controls and Rs. Furthermore, knockdown of Protocadherin alpha genes directly regulated iPSC-derived neurite length and morphology. Our results suggest that intrinsic differences in serotonergic neuron morphology and the resulting circuitry may contribute to SSRI resistance in MDD patients.

The social push for the therapeutic use of cannabis extracts has increased significantly over recent years. Cannabis is being used for treatment for conditions such as epilepsy, cancer and pain management. There are a range of medicinal cannabis products available, but the use of cannabis resin obtained by super critical fluid extraction, often diluted in oil, is becoming increasingly more prominent. Much of the research on cannabis has focused on plant biomass or the final therapeutic product with a concerning lack of information on the intermediate resin. This study aims to bridge the gap between current methods of analysis for biomass and the final therapeutic product by describing a fully developed and validated ultra-high-performance-liquid-chromatography method with diode array detection (UHPLC-DAD) for the qualification and quantification of the cannabinoids CBDA, CBD, CBN, THC, CBC and THCA, in medicinal cannabis biomass and resin obtained by super-critical fluid extraction (SFE). The method was validated for specificity, linearity, limit of detection (LOD), limit of quantitation (LOQ), precision, accuracy, robustness, spike recovery and stability in accordance with the Validation of Analytical Procedures: Text and Methodology Q2 to meet the requirements of the International Council for Harmonisation (ICH), Therapeutic Goods Authority (TGA) and the Food and Drug Administration (FDA) test method validation regulations.

Excess inflammation and canonical BMP receptor (BMPR) signaling are coinciding hallmarks of the early stages of injury-induced endochondral heterotopic ossification (EHO), especially in the rare genetic disease fibrodysplasia ossificans progressiva (FOP). Multiple inflammatory signaling pathways can synergistically enhance BMP-induced Smad1/5/8 activity in multiple cell types, suggesting the importance of pathway crosstalk in EHO and FOP. Toll-like receptors (TLRs) and IL-1 receptors mediate many of the earliest injury-induced inflammatory signals largely via MyD88-dependent pathways. Thus, the hypothesis that MyD88-dependent signaling is required for EHO was tested in vitro and in vivo using global or Pdgfrα-conditional deletion of MyD88 in FOP mice. As expected, IL-1β or LPS synergistically increased Activin A (ActA)-induced phosphorylation of Smad 1/5 in fibroadipoprogenitors (FAPs) expressing Alk2R206H. However, conditional deletion of MyD88 in Pdgfrα-positive cells of FOP mice did not significantly alter the amount of muscle injury-induced EHO. Even more surprisingly, injury-induced EHO was not significantly affected by global deletion of MyD88. These studies demonstrate that MyD88-dependent signaling is dispensable for injury-induced EHO in FOP mice.

Our study surveyed over 2000 employees of a community health care system in the Southeast United States for SARS-CoV2 antibodies. Survey included subjects' expectation of the result. Our local area had low community prevalence of SARS-CoV2 but low diagnostic testing capacity during much of the early phase of the epidemic. Despite only 3% positivity rate for antibodies in this population, 17% of subjects expected to have positive antibodies.

Liver-directed AAV transgene expression is enhanced by ImmTOR nanoparticles., Systemic AAV (adeno-associated virus) gene therapy is a promising approach for the treatment of inborn errors of metabolism, but questions remain regarding its potency and durability. Tolerogenic ImmTOR nanoparticles encapsulating rapamycin have been shown to block the formation of neutralizing anti-capsid antibodies, thereby enabling vector re-administration. Here, we further demonstrate that ImmTOR admixed with AAV vectors also enhances hepatic transgene expression at the initial dose of AAV vector, independent of its effects on adaptive immunity. ImmTOR enhances AAV trafficking to the liver, resulting in increased hepatic vector copy numbers and transgene mRNA expression. Enhanced transgene expression occurs through a mechanism independent of the AAV receptor and cannot be replicated in vivo with free rapamycin or empty nanoparticles. The multipronged mechanism of ImmTOR action makes it an attractive candidate to enable more efficient transgene expression at first dose while simultaneously inhibiting adaptive responses against AAV to enable repeat dosing.

Production and purification of a novel protein in plants results in the generation of multiple data sets leading to an optimized protocol for recovering the recombinant protein. This article presents the data collected in the process used to produce, purify and validate a catfish interleukin 22 (cfIL-22) expressed using a plant-based platform. A commonly used workflow for confirming optimal expression and extraction of the recombinant protein was employed and is outlined herein. The complete research article, including activity analysis of plant-produced cfIL-22, is published in Journal of Biotechnology Elkins and Dolan [1] . Data collected in optimizing the expression, purification and characterization process of cfIL-22 includes stained protein gels and western immunoblot analyses, DNA and protein sequencing, post-translational modification predictions and protein structure predictions. The value of this data lies not only in future work in expressing interleukin 22 orthologs but also provides a guide for optimizing the production and validating similar complex animal/human proteins produced in plants.

Many soil microorganisms have evolved catabolic strategies to utilize phenolic compounds arising from depolymerized lignin. We report the complete genome sequences of four Pseudomonas sp. isolates that demonstrated robust growth on a wide range of aromatic monomers and dimers that are relevant to the valorization of lignin into value-added chemicals.

Noctuid pests, including tobacco budworm (Chloridea virescens (Fab.)) and bollworm (Helicoverpa zea (Boddie)), are significant pests of southern row crops including cotton (Gossypium hirsutum L.), corn (Zea mays L.), and soybean (Glycine max (L.) Moench.). This pest complex is seasonally monitored through Hartstack traps that are baited with synthetic lepidopteran pheromones across the southern United States. We examined bycatch from the noctuid traps deployed across the Mississippi Delta in 2015, 2016, and 2017 for the presence of bees. The most abundant species collected were honey bees (Apis mellifera L.), bumble bees (Bombus spp.), and long-horned bees (Melissodes spp.); these three genera accounted for 82.4% of specimens collected. We also evaluated the proportion of local- and landscape-level habitats on the abundance and richness of the bees caught as bycatch. The proportion of natural and semi-natural habitat affected the abundance and richness of bees collected at the landscape level, but not at more local scales. Additional research is needed to better understand these interactions between bycatch and landscape factors to minimize non-target collections.

Rearing bollworm, Helicoverpa zea (Boddie), on synthetic diets for use in laboratory bioassays is a common practice around the world. Little is known about how insect diets currently utilized for bollworm rearing have been altered from their original nutrient composition and the implications this might have on insect fitness. Therefore, this study assessed notable shortcomings of a commonly used meridic diet by exposing bollworms to cotton (Gossypium hirsutum L) leaf tissue for short periods during early development to determine effect on larval and pupal survival and weights. Mean survival of larvae ranged from 60% when bollworms were exposed to Bt cotton leaves for 7 days to 100% on meridic diet. Mean pupation ranged from 58 to 97%, which closely mimicked survival of larvae at 7 days. Insect larvae reared on meridic diet through 7 days were fitter relative to those exposed to cotton leaf tissue, having greater larval weight and survival than other treatments. Conversely, pupal weight of bollworms exposed to meridic diet for the same 7-day period tended to lag behind those exposed to cotton leaf tissue. However, any fitness benefit gained from rearing larvae on leaf tissue might be offset by increased mortality before the pupal stage. The experiment demonstrated that larval and pupal fitness were impacted by supplementing meridic diet with leaf tissue, but additional research is needed to further investigate effects through multiple generations.

The soil- and seed-borne fungus, Fusarium oxysporum f. sp. vasinfectum race 4 (FOV4), causes Fusarium wilt in cotton (Gossypium spp.) and represents a significant threat to cotton production in the US. The objectives of this study were to enhance FOV4 resistance in heterogeneous Acala 1517-08 and its derivatives through three cycles of pedigree selection in 2018, 2019 and 2020 under FOV4-infested field conditions. In 2017, in a field not infested with FOV4, 46, 12, and 23 single plants were selected for productivity from a base population of 1100–1400 plants of Acala 1517-08 and its derived glandless lines, Acala 1517-18 GLS and NM 13P1125, respectively. In 2018, the single plant selections, controls and other lines were tested in progeny-rows in an FOV4 infested field for disease responses including mortality rate (MR), root vascular staining (RVS), and disease incidence (DI), resulting in 41, 15, and 26 single plants selected from progenies of the three lines, respectively, based on RVS. These single plant selections were tested in 82 progeny-rows in the same field in 2019, and the second selection cycle based on RVS advanced 40, 16, and 37 plants selected from the progenies of the three lines, respectively, for further evaluation and the third selection cycle in a progeny test in 2020. In 2020, an average of 81–84% plants died in highly susceptible controls, Pima S-7 and Pima DP 744, due to FOV4 infections, while the resistant controls, Pima S-6, Pima PHY 841 RF and NuMex COT 17 GLS, incurred up to 19–24% mortality. As a result of the three cycles of single plant selection, a total of 12 progenies were identified to be as resistant as the resistant controls. On the progeny basis, MR between years was significantly correlated, and MR at different evaluation dates was also significantly correlated with one another and with mean RVS and DI within year. However, due to possible escapes of plants from FOV4 infections, the original RVS scores of individual plant selections were not correlated with their respective progenies; and no significant differences were detected among progeny means grouped based on RVS ratings for single plant selections. The results demonstrate that early plant mortality is a superior means of selecting against FOV4 susceptibility, while using RVS rating late in the season if excluding seedling MR may inflate and distort the resistance level of genotypes, rendering it less reliable in assessing FOV4 resistance in cotton. In addition, many single plants selected for FOV4 resistance with no RVS may be escapes, necessitating repeated progeny tests and selection. The importance of pedigree selection within germplasm, choice of resistant and susceptible checks, FOV4 inoculum density, and the use of RVS, and resistance versus tolerance in response to FOV4 infections under field conditions are discussed in detail.

Controlled environment crop production recommendations often use the daily light integral (DLI) to quantify the light requirements of specific crops. Sole-source electric lighting, used in plant factories, and supplemental electric lighting, used in greenhouses, may be required to attain a specific DLI. Electric lighting is wasteful if not provided in a way that promotes efficient photochemistry. The quantum yield of photosystem II (&Phi, PSII), the fraction of absorbed light used for photochemistry, decreases with increasing photosynthetic photon flux density (PPFD). Thus, we hypothesized that the daily photochemical integral (DPI), the total electron transport through photosystem II (PSII) integrated over 24 h, would increase if the same DLI was provided at a lower PPFD over a longer photoperiod. To test this, &Phi, PSII and the electron transport rate (ETR) of lettuce (Lactuca sativa &lsquo, Green Towers&rsquo, ) were measured in a growth chamber at DLIs of 15 and 20 mol m&minus, 2 d&minus, 1 over photoperiods ranging from 7 to 22 h. This resulted in PPFDs of 189 to 794 &mu, mol m&minus, 2 s&minus, 1. The &Phi, PSII decreased from 0.67 to 0.28 and ETR increased from 55 to 99 &mu, 1 as PPFD increased from 189 to 794 &mu, 1. The DPI increased linearly as the photoperiod increased, but the magnitude of this response depended on DLI. With a 7-h photoperiod, the DPI was &asymp, 2.7 mol m&minus, 1, regardless of DLI. However, with a 22-h photoperiod, the DPI was 4.54 mol m&minus, 1 with a DLI of 15 mol m&minus, 1 and 5.78 mol m&minus, 1 with a DLI of 20 mol m&minus, 1. Our hypothesis that DPI can be increased by providing the same DLI over longer photoperiods was confirmed.

Production of IFN-γ is a key innate immune mechanism that limits replication of intracellular bacteria such as Francisella tularensis (Ft) until adaptive immune responses develop. Previously, we demonstrated that the host cell types responsible for IFN-γ production in response to murine Francisella infection include not only natural killer (NK) and T cells, but also a variety of myeloid cells. However, production of IFN-γ by mouse dendritic cells (DC) is controversial. Here, we directly demonstrated substantial production of IFN-γ by DC, as well as hybrid NK-DC, from LVS-infected wild type C57BL/6 or Rag1 knockout mice. We demonstrated that the numbers of conventional DC producing IFN-γ increased progressively over the course of 8 days of LVS infection. In contrast, the numbers of conventional NK cells producing IFN-γ, which represented about 40% of non-B/T IFN-γ-producing cells, peaked at day 4 after LVS infection and declined thereafter. This pattern was similar to that of hybrid NK-DC. To further confirm IFN-γ production by infected cells, DC and neutrophils were sorted from naive and LVS-infected mice and analyzed for gene expression. Quantification of LVS by PCR revealed the presence of Ft DNA not only in macrophages, but also in highly purified, IFN-γ producing DC and neutrophils. Finally, production of IFN-γ by infected DC was confirmed by immunohistochemistry and confocal microscopy. Notably, IFN-γ production patterns similar to those in wild type mice were observed in cells derived from LVS-infected TLR2, TLR4, and TLR2xTLR9 knockout (KO) mice, but not from MyD88 KO mice. Taken together, these studies demonstrate the pivotal roles of DC and MyD88 in IFN-γ production and in initiating innate immune responses to this intracellular bacterium.

There is increasing evidence of a significant correlation between prolonged drug-target residence time and increased drug efficacy. Here, we report a structural rationale for kinetic selectivity between two closely related kinases: focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2). We found that slowly dissociating FAK inhibitors induce helical structure at the DFG motif of FAK but not PYK2. Binding kinetic data, high-resolution structures and mutagenesis data support the role of hydrophobic interactions of inhibitors with the DFG-helical region, providing a structural rationale for slow dissociation rates from FAK and kinetic selectivity over PYK2. Our experimental data correlate well with computed relative residence times from molecular simulations, supporting a feasible strategy for rationally optimizing ligand residence times. We suggest that the interplay between the protein structural mobility and ligand-induced effects is a key regulator of the kinetic selectivity of inhibitors of FAK versus PYK2.

Francisella tularensis is a highly infectious intracellular bacterium that causes tularemia by invading and replicating in mammalian myeloid cells. Francisella primarily invades host macrophages, where it escapes phagosomes within a few hours and replicates in the cytoplasm. Less is known about how Francisella traffics within macrophages or exits into the extracellular environment for further infection. Immune T lymphocytes control the replication of Francisella within macrophages in vitro by a variety of mechanisms, but nothing is known about intracellular bacterial trafficking in the face of such immune pressure. Here we used a murine model of infection with a Francisella attenuated live vaccine strain (LVS), which is under study as a human vaccine, to evaluate the hypothesis that immune T cells control intramacrophage bacterial growth by re-directing bacteria into toxic intracellular compartments of infected macrophages. We visualized the interactions of lymphocytes and LVS-infected macrophages using confocal microscopy and characterized LVS intramacrophage trafficking when co-cultured with immune lymphocytes. We focused on the late stages of infection after bacteria escape from phagosomes, through bacterial replication and the death of macrophages. We found that the majority of LVS remained cytosolic in the absence of immune pressure, eventually resulting in macrophage death. In contrast, co-culture of LVS-infected macrophages with LVS-immune lymphocytes halted LVS replication and inhibited the spread of LVS infection between macrophages, but bacteria did not return to vacuoles such as lysosomes or autophagosomes and macrophages did not die. Therefore, immune lymphocytes directly limit intracellular bacterial replication within the cytoplasm of infected macrophages.

Kinases represent one of the most intensively pursued groups of targets in modern-day drug discovery. Often it is desirable to achieve selective inhibition of the kinase of interest over the remaining ∼500 kinases in the human kinome. This is especially true when inhibitors are intended to be used to study the biology of the target of interest. We present a pipeline of open-source software that analyzes public domain data to repurpose compounds that have been used in previous kinase inhibitor development projects. We define the dual-specificity tyrosine-regulated kinase 1A (DYRK1A) as the kinase of interest, and by addition of a single methyl group to the chosen starting point we remove glycogen synthase kinase β (GSK3β) and cyclin-dependent kinase (CDK) inhibition. Thus, in an efficient manner we repurpose a GSK3β/CDK chemotype to deliver 8b, a highly selective DYRK1A inhibitor.

An increased incidence of dilated cardiomyopathy and atrial thrombosis was noted in a breeding colony of BALB/c mice deficient in IL4 receptor α. The condition affected mice of both sexes and of various ages, and extensive testing (microbiology, serology, histopathology) failed to ascertain the cause. Transmission electron microscopy of heart samples showed structural defects in the myocardial intercalated disks, characterized by unorganized and heavily convoluted arrangement with lower density and less prominent desmosomes and adherens junctions, widening of the intercellular space, myofibrillar lysis adjacent to intercalated disks, occasional sarcomere lysis with marked myofiber degeneration, vacuolation, accumulation of cell debris, and myelin figures. The intercalated disk contains cell adhesion molecules that form cell junctions, allowing contraction coupling of cardiomyocytes and the electrical and mechanical connection between cardiac fibers. Thus, defects at this level result in poor myocardial contraction, intracardiac blood stagnation, and consequently cardiac dilation with clinical signs of heart failure. The background strain or, potentially, the Cre–loxP-mediated recombination system used to create these mice may have contributed to the elevated incidence of cardiomyopathy and atrial thrombosis in this colony. Due to the backcrossing breeding scheme used, we cannot discount the emergence and colonywide dissemination of a spontaneous mutation that affects the intercalated disk. This report underscores the importance of carefully monitoring genetically modified mice colonies for unexpected phenotypes that may result from spontaneous or unintended mutations or enhanced strain background pathology.

We vaccinated the Diversity Outbred (DO) population of mice with BCG, the only vaccine currently used to protect against tuberculosis, and then challenged them with M. tuberculosis by aerosol. We found that the BCG-vaccinated DO mouse population exhibited a wide range of outcomes, in which outcomes in individual mice ranged from minimal respiratory or systemic disease to fulminant disease and death. The breadth of these outcomes appears similar to the range seen in people, indicating that DO mice may serve as an improved small-animal model to study tuberculosis infection and immunity. Moreover, sophisticated tools are available for the use of these mice to map genes contributing to control of vaccination. Thus, the present studies provided an important new tool in the fight against tuberculosis., Many studies of Mycobacterium tuberculosis infection and immunity have used mouse models. However, outcomes of vaccination and challenge with M. tuberculosis in inbred mouse strains do not reflect the full range of outcomes seen in people. Previous studies indicated that the novel Diversity Outbred (DO) mouse population exhibited a spectrum of outcomes after primary aerosol infection with M. tuberculosis. Here, we demonstrate the value of this novel mouse population for studies of vaccination against M. tuberculosis aerosol challenge. Using the only currently licensed tuberculosis vaccine, we found that the DO population readily controlled systemic Mycobacterium bovis BCG bacterial burdens and that BCG vaccination significantly improved survival across the DO population upon challenge with M. tuberculosis. Many individual DO mice that were vaccinated with BCG and then challenged with M. tuberculosis exhibited low bacterial burdens, low or even no systemic dissemination, little weight loss, and only minor lung pathology. In contrast, some BCG-vaccinated DO mice progressed quickly to fulminant disease upon M. tuberculosis challenge. Across the population, most of these disease parameters were at most modestly correlated with each other and were often discordant. This result suggests the need for a multiparameter metric to better characterize “disease” and “protection,” with closer similarity to the complex case definitions used in people. Taken together, these results demonstrate that DO mice provide a novel small-animal model of vaccination against tuberculosis that better reflects the wide spectrum of outcomes seen in people. IMPORTANCE We vaccinated the Diversity Outbred (DO) population of mice with BCG, the only vaccine currently used to protect against tuberculosis, and then challenged them with M. tuberculosis by aerosol. We found that the BCG-vaccinated DO mouse population exhibited a wide range of outcomes, in which outcomes in individual mice ranged from minimal respiratory or systemic disease to fulminant disease and death. The breadth of these outcomes appears similar to the range seen in people, indicating that DO mice may serve as an improved small-animal model to study tuberculosis infection and immunity. Moreover, sophisticated tools are available for the use of these mice to map genes contributing to control of vaccination. Thus, the present studies provided an important new tool in the fight against tuberculosis.

Most livestock metabolomic studies involve relatively small, homogenous populations of animals. However, livestock farming systems are non-homogenous, and large and more diverse datasets are required to ensure that biomarkers are robust. The aims of this study were therefore to (1) investigate the feasibility of using a large and diverse dataset for untargeted proton nuclear magnetic resonance (1H NMR) serum metabolomic profiling, and (2) investigate the impact of fixed effects (farm of origin, parity and stage of lactation) on the serum metabolome of early-lactation dairy cows. First, we used multiple linear regression to correct a large spectral dataset (707 cows from 13 farms) for fixed effects prior to multivariate statistical analysis with principal component analysis (PCA). Results showed that farm of origin accounted for up to 57% of overall spectral variation, and nearly 80% of variation for some individual metabolite concentrations. Parity and week of lactation had much smaller effects on both the spectra as a whole and individual metabolites (&lt, 3% and &lt, 20%, respectively). In order to assess the effect of fixed effects on prediction accuracy and biomarker discovery, we used orthogonal partial least squares (OPLS) regression to quantify the relationship between NMR spectra and concentrations of the current gold standard serum biomarker of energy balance, &beta, hydroxybutyrate (BHBA). Models constructed using data from multiple farms provided reasonably robust predictions of serum BHBA concentration (0.05 &le, RMSE &le, 0.18). Fixed effects influenced the results biomarker discovery, however, these impacts could be controlled using the proposed method of linear regression spectral correction.

Over one hundred bacterial species have been determined to comprise the human microbiota in a healthy individual. Bacteria including Escherichia coli, Bacillus cereus, Clostridioides difficile, and Vibrio parahaemolyticus are found inside of the human body and B. cereus and E. coli are also found on the skin. These bacteria can act as human pathogens upon ingestion of contaminated food or water, if they enter an open wound, or antibiotics, and environment or stress can alter the microbiome. In this study, we present new polymerase chain reaction (PCR) high-resolution melt (HRM) assays to detect and identify the above microorganisms. Amplified DNA from C. difficile, E. coli, B. cereus, and V. parahaemolyticus melted at 80.37 &plusmn, 0.45 &deg, C, 82.15 &plusmn, 0.37 &deg, C, 84.43 &plusmn, 0.50 &deg, C, and 86.74 &plusmn, 0.65 &deg, C, respectively. A triplex PCR assay was developed to simultaneously detect and identify E. coli, B. cereus, and V. parahaemolyticus, and cultured microorganisms were successfully amplified, detected, and identified. The assays demonstrated sensitivity, specificity, reproducibility, and robustness in testing.

Alterations in the NRF2/KEAP1 pathway result in the constitutive activation of NRF2, leading to the aberrant induction of antioxidant and detoxification enzymes, including NQO1. The NQO1 bioactivatable agent β-lapachone can target cells with high NQO1 expression but relies in the generation of reactive oxygen species (ROS), which are actively scavenged in cells with NRF2/KEAP1 mutations. However, whether NRF2/KEAP1 mutations influence the response to β-lapachone treatment remains unknown. To address this question, we assessed the cytotoxicity of β-lapachone in a panel of NSCLC cell lines bearing either wild-type or mutant KEAP1. We found that, despite overexpression of NQO1, KEAP1 mutant cells were resistant to β-lapachone due to enhanced detoxification of ROS, which prevented DNA damage and cell death. To evaluate whether specific inhibition of the NRF2-regulated antioxidant enzymes could abrogate resistance to β-lapachone, we systematically inhibited the four major antioxidant cellular systems using genetic and/or pharmacologic approaches. We demonstrated that inhibition of the thioredoxin-dependent system or copper-zinc superoxide dismutase (SOD1) could abrogate NRF2-mediated resistance to β-lapachone, while depletion of catalase or glutathione was ineffective. Interestingly, inhibition of SOD1 selectively sensitized KEAP1 mutant cells to β-lapachone exposure. Our results suggest that NRF2/KEAP1 mutational status might serve as a predictive biomarker for response to NQO1-bioactivatable quinones in patients. Further, our results suggest SOD1 inhibition may have potential utility in combination with other ROS inducers in patients with KEAP1/NRF2 mutations., Graphical abstract Image 1, Highlights • Aberrant activation of NRF2 in non-small cell lung cancer promotes resistance to β-lapachone via the antioxidant defense. • Inhibition of the thioredoxin-dependent system and superoxide dismutase 1 increase sensitivity to β-lapachone treatment. • Mutations in the NRF2/KEAP1 pathway might serve as predictive biomarker for response to β-lapachone in patients.

Development of grass–endophyte associations with minimal or no detrimental effects in combination with beneficial characteristics is important for pastoral agriculture. The feasibility of enhancing production of an endophyte-derived beneficial alkaloid through introduction of an additional gene copy was assessed in a proof-of-concept study. Sexual and asexual Epichloë species that form symbiotic associations with cool-season grasses of the Poaceae sub-family Pooideae produce bioactive alkaloids that confer resistance to herbivory by a number of organisms. Of these, peramine is thought to be crucial for protection of perennial ryegrass (Lolium perenne L.) from the Argentinian stem weevil, an economically important exotic pest in New Zealand, contributing significantly to pasture persistence. A single gene (perA) has been identified as solely responsible for peramine biosynthesis and is distributed widely across Epichloë taxa. In the present study, a functional copy of the perA gene was introduced into three recipient endophyte genomes by Agrobacterium tumefaciens-mediated transformation. The target strains included some that do not produce peramine, and others containing different perA gene copies. Mitotically stable transformants generated from all three endophyte strains were able to produce peramine in culture and in planta at variable levels. In summary, this study provides an insight into the potential for artificial combinations of alkaloid biosynthesis in a single endophyte strain through transgenesis, as well as the possibility of using novel genome editing techniques to edit the perA gene of non-peramine producing strains.

We describe the assembly and annotation of a chemogenomic set of protein kinase inhibitors as an open science resource for studying kinase biology. The set only includes inhibitors that show potent kinase inhibition and a narrow spectrum of activity when screened across a large panel of kinase biochemical assays. Currently, the set contains 187 inhibitors that cover 215 human kinases. The kinase chemogenomic set (KCGS) is the most highly annotated set of selective kinase inhibitors available to researchers for use in cell-based screens.

Due to widespread stocking, Rainbow Trout (Oncorhynchus mykiss Walbaum) are perhaps the most widely distributed invasive species in the world. Nonetheless, little is known about the effects of stocked Rainbow Trout on native non‐game species. We conducted experiments in an artificial stream to assess the effects of hatchery Rainbow Trout on home range and behaviour of Warpaint Shiners (Luxilus coccogenis Cope), a common minnow frequently found in stocked Southern Appalachian streams. We used the LoCoH algorithm to generate polygons describing the home ranges used by Warpaint Shiners. When a stocked trout was present Warpaint Shiners: (a) increased home range size by 57%, (b) were displaced into higher velocity mesohabitats, and (c) reduced mean overlap between the home ranges of individual warpaint shiners. Rainbow Trout did not significantly affect the edge/area ratio of Warpaint Shiner home ranges. Warpaint Shiner density (two and five fish treatments) did not significantly affect any response variable. Displacement from preferred microhabitats and increases in home range size likely result in increased energy expenditure and exposure to potential predators (i.e., decreased individual fitness) of Warpaint Shiners when stocked trout are present.

The U.S. Food and Drug Administration Escherichia coli Identification (FDA-ECID) microarray provides rapid molecular characterization of E. coli. The effectiveness of the FDA-ECID for characterizing Shiga toxin–producing E. coli (STEC) was evaluated by three federal laboratories and one reference laboratory with a panel of 54 reference E. coli strains from the External Quality Assurance program. Strains were tested by FDA-ECID for molecular serotyping (O and H antigens), Shiga toxin subtyping, and the presence of the ehxA and eae genes for enterohemolysin and intimin, respectively. The FDA-ECID O typing was 96% reproducible among the four laboratories and 94% accurate compared with the reference External Quality Assurance data. Discrepancies were due to the absence of O41 target loci on the array and to two pairs of O types with identical target sequences. H typing was 96% reproducible and 100% accurate, with discrepancies due to two strains from one laboratory that were identified as mixed by FDA-ECID. Shiga toxin (Stx) type 1 subtyping was 100% reproducible and accurate, and Stx2 subtyping was 100% reproducible but only 64% accurate. FDA-ECID identified most Stx2 subtypes but had difficulty distinguishing among stx2a, stx2c, and stx2d genes because of close similarities of these sequences. FDA-ECID was 100% effective for detecting ehxA and eae and accurately subtyped the eae alleles. This interlaboratory study revealed that FDA-ECID for STEC characterization was highly reproducible for molecular serotyping, stx and eae subtyping, and ehxA detection. However, the array was less useful for distinguishing among the highly homologous O antigen genes and the stx2a, stx2c, and stx2d subtypes.

SRA Dose-Response and Microbial Risk Analysis Specialty Groups jointly sponsored symposia that addressed the intersections between the "microbiome revolution" and dose response. Invited speakers presented on innovations and advances in gut and nasal microbiota (normal microbial communities) in the first decade after the Human Microbiome Project began. The microbiota and their metabolites are now known to influence health and disease directly and indirectly, through modulation of innate and adaptive immune systems and barrier function. Disruption of healthy microbiota is often associated with changes in abundance and diversity of core microbial species (dysbiosis), caused by stressors including antibiotics, chemotherapy, and disease. Nucleic-acid-based metagenomic methods demonstrated that the dysbiotic host microbiota no longer provide normal colonization resistance to pathogens, a critical component of innate immunity of the superorganism. Diverse pathogens, probiotics, and prebiotics were considered in human and animal models (in vivo and in vitro). Discussion included approaches for design of future microbial dose-response studies to account for the presence of the indigenous microbiota that provide normal colonization resistance, and the absence of the protective microbiota in dysbiosis. As NextGen risk analysis methodology advances with the "microbiome revolution," a proposed new framework, the Health Triangle, may replace the old paradigm based on the Disease Triangle (focused on host, pathogen, and environment) and germophobia. Collaborative experimental designs are needed for testing hypotheses about causality in dose-response relationships for pathogens present in our environments that clearly compete in complex ecosystems with thousands of bacterial species dominating the healthy superorganism.

Virtually all the red blood cell and platelet antigen systems have been characterized at the molecular level. Highly reliable methods for red blood cell and platelet antigen genotyping are now available. Genotyping is a useful adjunct to traditional serology and can help resolve complex serologic problems. Although red blood cell and platelet phenotypes can be inferred from genotype, knowledge of the molecular basis is essential for accurate assignment. Genotyping of blood donors is an effective method of identifying antigen-negative and/or particularly rare donors. Cell-free DNA analysis provides a promising noninvasive method of assessing fetal genotypes of blood group alloantigens.

Background We report whether the etiology underlying associations of childhood ADHD with adolescent alcohol and marijuana involvement is consistent with causal relationships or shared predispositions, and whether it differs by gender. Methods In three population-based twin samples (N = 3762; 64% monozygotic), including one oversampling females with ADHD, regressions were conducted with childhood inattentive or hyperactive-impulsive symptoms predicting alcohol and marijuana outcomes by age 17. To determine whether ADHD effects were consistent with causality, twin difference analyses divided effects into those shared between twins in the pair and those differing within pairs. Results Adolescents with more severe childhood ADHD were more likely to initiate alcohol and marijuana use earlier, escalate to frequent or heavy use, and develop symptoms. While risks were similar across genders, females with more hyperactivity-impulsivity had higher alcohol consumption and progressed further toward daily marijuana use than did males. Monozygotic twins with more severe ADHD than their co-twins did not differ significantly on alcohol or marijuana outcomes, however, suggesting a non-causal relationship. When co-occurring use of other substances and conduct/oppositional defiant disorders were considered, hyperactivity-impulsivity remained significantly associated with both substances, as did inattention with marijuana, but not alcohol. Conclusions Childhood ADHD predicts when alcohol and marijuana use are initiated and how quickly use escalates. Shared familial environment and genetics, rather than causal influences, primarily account for these associations. Stronger relationships between hyperactivity-impulsivity and heavy drinking/frequent marijuana use among adolescent females than males, as well as the greater salience of inattention for marijuana, merit further investigation.

Psilocybe cubensis,or“magic mushroom,” is the most common species of fungus with psychedelic characteristics. Two primer sets were designed to target Psilocybe DNA using web-based software and NBCI gene sequences. DNA was extracted from eighteen samples, including twelve mushroom species, using the Qiagen DNeasy Plant Mini Kit. The DNA was amplified by the polymerase chain reaction (PCR) using the primers and a master mix containing either a SYBR Green I, RadiantTM Green, or LCGreen Plus intercalating dye; amplicon size was determined using agarose gel electrophoresis. The PCR assays were tested for amplifiability, specificity, reproducibility, robustness, sensitivity, and multiplexing with primers that target marijuana. The observed high resolution melt (HRM) temperatures for primer sets 1 and 7 were 78.85 +_ 0.31°C and 73.22 +- 0.61°C, respectively, using SYBR Green I dye and 81.67+- 0.06°C and 76.04 +- 0.11°C, respectively, using RadiantTM Green dye.

A wealth of clustering algorithms are available for single-cell RNA sequencing (scRNA-seq) data to enable the identification of functionally distinct subpopulations that each possess a different pattern of gene expression activity. Implementation of these methods requires a choice of resolution parameter to determine the number of clusters, and critical judgment from the researchers is required to determine the desired resolution. This supervised process takes significant time and effort. Moreover, it can be difficult to compare and characterize the evolution of cell clusters from results obtained at one single resolution. To overcome these challenges, we built Multi-resolution Reconciled Tree (MRtree), a highly flexible tree-construction algorithm that generates a cluster hierarchy from flat clustering results attained for a range of resolutions. Because MRtree can be coupled with most scRNA-seq clustering algorithms, it inherits the robustness and versatility of a flat clustering approach, while maintaining the hierarchical structure of cells. The constructed trees from multiple scRNA-seq datasets effectively reflect the extent of transcriptional distinctions among cell groups and align well with levels of functional specializations among cells. Importantly, application to fetal brain cells identified subtypes of cells determined mainly by maturation states, spatial location and terminal specification.

Respiratory infection of mice with Francisella novicida has recently been used as a model for the highly virulent human pathogen Francisella tularensis. Similar to F. tularensis, even small doses of F. novicida administered by respiratory routes are lethal for inbred laboratory mice. This feature obviously limits study of infection-induced immunity. Parenteral sublethal infections of mice with F. novicida are feasible, but the resulting immune responses are incompletely characterized. Here we use parenteral intradermal (i.d.) and intraperitoneal (i.p.) F. novicida infections of C57BL/6J mice to determine the role of B cells in controlling primary and secondary F. novicida infections. Despite developing comparable levels of F. novicida-primed T cells, B cell knockout mice were much more susceptible to both primary i.d. infection and secondary i.p. challenge than wild type (normal) C57BL/6J mice. Transfer of F. novicida-immune sera to either wild type C57BL/6J mice or to B cell knockout mice did not appreciably impact survival of subsequent lethal F. novicida challenge. However, F. novicida-immune mice that were depleted of T cells after priming but just before challenge survived and cleared secondary i.p. F. novicida challenge. Collectively these results indicate that B cells, if not serum antibodies, play a major role in controlling F. novicida infections in mice.