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Oxygen isotope (δ18O) sclerochronology of benthic marine molluscs provides a means of reconstructing the seasonal range in seafloor temperature, subject to use of an appropriate equation relating shell δ18O to temperature and water δ18O, a reasonably accurate estimation of water δ18O, and due consideration of growth-rate effects. Taking these factors into account, δ18O data from late Pliocene bivalves of the southern North Sea basin (Belgium and the Netherlands) indicate a seasonal seafloor range a little smaller than now in the area. Microgrowth-increment data from Aequipecten opercularis, together with the species composition of the bivalve assemblage and aspects of preservation, suggest a setting below the summer thermocline for all but the latest material investigated. This implies a higher summer temperature at the surface than on the seafloor and consequently a greater seasonal range. A reasonable (3 ∘C) estimate of the difference between maximum seafloor and surface temperature under circumstances of summer stratification points to seasonal surface ranges in excess of the present value (12.4 ∘C nearby). Using a model-derived estimate of water δ18O (0.0 ‰), summer surface temperature was initially in the cool temperate range ( ∘C) and then (during the Mid-Piacenzian Warm Period; MPWP) increased into the warm temperate range (>20 ∘C) before reverting to cool temperate values (in conjunction with shallowing and a loss of summer stratification). This pattern is in agreement with biotic-assemblage evidence. Winter temperature was firmly in the cool temperate range ( ∘C) throughout, contrary to previous interpretations. Averaging of summer and winter surface temperatures for the MPWP provides a figure for annual sea surface temperature that is 2–3 ∘C higher than the present value (10.9 ∘C nearby) and in close agreement with a figure obtained by averaging alkenone and TEX86 temperatures for the MPWP from the Netherlands. These proxies, however, respectively, underestimate summer temperature and overestimate winter temperature, giving an incomplete picture of seasonality. A higher annual temperature than now is consistent with the notion of global warmth in the MPWP, but a low winter temperature in the southern North Sea basin suggests regional reduction in oceanic heat supply, contrasting with other interpretations of North Atlantic oceanography during the interval. Carbonate clumped isotope (Δ47) and biomineral unit thermometry offer means of checking the δ18O-based temperatures.

Patients with established rheumatoid arthritis (RA) demonstrate altered immune responses to Epstein-Barr virus (EBV), but the presence and roles of EBV have not been fully explored during the pre-clinical disease period. This study was undertaken to determine if EBV infection, as evidenced by an altered anti-EBV antibody response, either plays an important role in driving the development of RA or is a result of expanded RA-related autoimmunity.A total of 83 subjects with RA according to the 1987 American College of Rheumatology (ACR) criteria and 83 age-, sex-, and race-matched control subjects without RA were included in our study. We collected sera from RA subjects and matched controls during the pre-RA and post-RA diagnosis periods and tested the sera for the presence of 5 anti-EBV antibodies (anti-EBV nuclear antigen 1 IgG isotype, anti-viral capsid antigen [anti-VCA] isotypes IgG and IgA, and anti-early antigen [EA] isotypes IgG and IgA), 7 RA-related autoantibodies (rheumatoid factor measured by nephelometry [RF-Neph] as well as isotype-specific IgA-RF, IgM-RF, and IgG-RF, and anti-cyclic citrullinated peptide [anti-CCP] antibodies, including anti-CCP2, anti-CCP3, and anti-CCP3.1), 22 cytokines/chemokines, 36 individual anti-citrullinated protein antibodies, and IgG-cytomegalovirus (CMV) antibodies. Random forest classification, mixed modeling, and joint mixed modeling were used to determine differences in anti-EBV antibody levels between RA subjects and controls.Random forest analysis identified the presence of preclinical EBV antibodies in the serum that differentiated RA subjects from controls without RA. Specifically, IgG-EA antibody levels were higher in RA subjects (mean ± SD 0.82 ± 0.72 international standardized ratio [ISR]) compared to controls (mean ± SD 0.49 ± 0.28 ISR). In subjects with RA, elevated serum IgG-EA levels in the preclinical period before seroconversion were significantly correlated with increased serum IgM-RF levels (P = 0.007), whereas this correlation was not seen in control subjects without RA (P = 0.15). IgG-CMV antibody levels did not differ between groups.Subjects whose serum IgG-EA antibody levels are elevated in the preclinical period will eventually develop RA, which suggests that EBV reactivation cycles are increased during the preclinical period of RA. A combination of RF and EBV reactivation may play an important role in the development of RA.

Secondary cell walls (SCWs) in stem xylem vessel and fibre cells enable plants to withstand the enormous compressive forces associated with upright growth. It remains unclear if xylem vessel and fibre cells can directly sense mechanical stimuli and modify their SCW during development. We provide evidence that Arabidopsis SCW-specific Fasciclin-Like Arabinogalactan-proteins 11 (FLA11) and 12 (FLA12) are possible cell surface sensors regulating SCW development in response to mechanical stimuli. Plants overexpressing FLA11 (OE-FLA11) showed earlier SCW development compared to the wild-type (WT) and altered SCW properties that phenocopy WT plants under compression stress. By contrast, OE-FLA12 stems showed higher cellulose content compared to WT plants, similar to plants experiencing tensile stress. fla11, OE-FLA11, fla12, and OE-FLA12 plants showed altered SCW responses to mechanical stress compared to the WT. Quantitative polymerase chain reaction (qPCR) and RNA-seq analysis revealed the up-regulation of genes and pathways involved in stress responses and SCW synthesis and regulation. Analysis of OE-FLA11 nst1 nst3 plants suggests that FLA11 regulation of SCWs is reliant on classical transcriptional networks. Our data support the involvement of FLA11 and FLA12 in SCW sensing complexes to fine-tune both the initiation of SCW development and the balance of lignin and cellulose synthesis/deposition in SCWs during development and in response to mechanical stimuli.

Simple Summary Targeting dysregulated cellular metabolism is a promising avenue to treat metastatic disease. The aim of our study was to identify genes downstream of the hypoxia-inducible factor (HIF)-1 transcription factor that are amenable to therapeutic intervention to treat metastatic breast cancer (MBC). We identified creatine kinase, brain isoform (CKB) as an HIF-dependent gene that strongly promotes invasion and metastasis in estrogen-receptor (ER)-negative breast cancer models. Deletion of Ckb also repressed glycolysis and mitochondrial respiration, leading to a reduction in intracellular ATP. Either the deletion of Ckb or inhibition of creatine kinase (CK) activity using the creatine analog cyclocreatine (cCr) repressed cell invasion, the formation of invadopodia and lung metastasis. In addition, when paired with paclitaxel or doxorubicin, cCr enhanced growth inhibition in an additive or synergistic manner. cCr may be an effective anti-metastatic agent in ER-negative, HIF-1α-positive breast cancers, targeting both cellular metabolism and motility, particularly when paired with conventional cytotoxic agents. Abstract The oxygen-responsive hypoxia inducible factor (HIF)-1 promotes several steps of the metastatic cascade. A hypoxic gene signature is enriched in triple-negative breast cancers (TNBCs) and is correlated with poor patient survival. Inhibiting the HIF transcription factors with small molecules is challenging; therefore, we sought to identify genes downstream of HIF-1 that could be targeted to block invasion and metastasis. Creatine kinase brain isoform (CKB) was identified as a highly differentially expressed gene in a screen of HIF-1 wild type and knockout mammary tumor cells derived from a transgenic model of metastatic breast cancer. CKB is a cytosolic enzyme that reversibly catalyzes the phosphorylation of creatine, generating phosphocreatine (PCr) in the forward reaction, and regenerating ATP in the reverse reaction. Creatine kinase activity is inhibited by the creatine analog cyclocreatine (cCr). Loss- and gain-of-function genetic approaches were used in combination with cCr therapy to define the contribution of CKB expression or creatine kinase activity to cell proliferation, migration, invasion, and metastasis in ER-negative breast cancers. CKB was necessary for cell invasion in vitro and strongly promoted tumor growth and lung metastasis in vivo. Similarly, cyclocreatine therapy repressed cell migration, cell invasion, the formation of invadopodia and lung metastasis. Moreover, in common TNBC cell line models, the addition of cCr to conventional cytotoxic chemotherapy agents was either additive or synergistic to repress tumor cell growth.

The dynein motor protein complex is required for retrograde transport but the functions of the intermediate-light chains that form the cargo-binding complex are not elucidated and the importance of individual subunits in maintaining cellular homeostasis is unknown. Here, using mRNA arrays and protein analysis, we show that the dynein subunit, DYNC1LI2 (dynein, cytoplasmic 1 light intermediate chain 2) is downregulated in cystinosis, a lysosomal storage disorder caused by genetic defects in CTNS (cystinosin, lysosomal cystine transporter). Reconstitution of DYNC1LI2 expression in ctns-/- cells reestablished endolysosomal dynamics. Defective vesicular trafficking in cystinotic cells was rescued by DYNC1LI2 expression which correlated with decreased endoplasmic reticulum stress manifested as decreased expression levels of the chaperone HSPA5/GRP78, and the transcription factors ATF4 and DDIT3/CHOP. Mitochondrial fragmentation, membrane potential and endolysosomal-mitochondrial association in cystinotic cells were rescued by DYNC1LI2. Survival of cystinotic cells to oxidative stress was increased by DYNC1LI2 reconstitution but not by its paralog DYNC1LI1, which also failed to decrease ER stress and mitochondrial fragmentation. DYNC1LI2 expression rescued the localization of the chaperone-mediated autophagy (CMA) receptor LAMP2A, CMA activity, cellular homeostasis and LRP2/megalin expression in cystinotic proximal tubule cells, the primary cell type affected in cystinosis. DYNC1LI2 failed to rescue phenotypes in cystinotic cells when LAMP2A was downregulated or when co-expressed with dominant negative (DN) RAB7 or DN-RAB11, which impaired LAMP2A trafficking. DYNC1LI2 emerges as a regulator of cellular homeostasis and potential target to repair underlying trafficking and CMA in cystinosis, a mechanism that is not restored by lysosomal cystine depletion therapies.Abbreviations: ACTB: actin, beta; ATF4: activating transcription factor 4; CMA: chaperone-mediated autophagy; DYNC1LI1: dynein cytoplasmic 1 light intermediate chain 1; DYNC1LI2: dynein cytoplasmic 1 light intermediate chain 2; ER: endoplasmic reticulum; LAMP1: lysosomal associated membrane protein 1; LAMP2A: lysosomal associated membrane protein 2A; LIC: light-intermediate chains; LRP2/Megalin: LDL receptor related protein 2; PTCs: proximal tubule cells; RAB: RAB, member RAS oncogene family; RAB11FIP3: RAB11 family interacting protein 3; RILP: Rab interacting lysosomal protein.

BackgroundZebrafish pigment cell differentiation provides an attractive model for studying cell fate progression as a neural crest progenitor engenders diverse cell types, including two morphologically distinct pigment cells: black melanophores and reflective iridophores. Nontrivial classical genetic and transcriptomic approaches have revealed essential molecular mechanisms and gene regulatory circuits that drive neural crest-derived cell fate decisions. However, how the epigenetic landscape contributes to pigment cell differentiation, especially in the context of iridophore cell fate, is poorly understood.ResultsWe chart the global changes in the epigenetic landscape, including DNA methylation and chromatin accessibility, during neural crest differentiation into melanophores and iridophores to identify epigenetic determinants shaping cell type-specific gene expression. Motif enrichment in the epigenetically dynamic regions reveals putative transcription factors that might be responsible for driving pigment cell identity. Through this effort, in the relatively uncharacterized iridophores, we validatealx4aas a necessary and sufficient transcription factor for iridophore differentiation and present evidence onalx4a’s potential regulatory role in guanine synthesis pathway.ConclusionsPigment cell fate is marked by substantial DNA demethylation events coupled with dynamic chromatin accessibility to potentiate gene regulation through cis-regulatory control. Here, we provide a multi-omic resource for neural crest differentiation into melanophores and iridophores. This work led to the discovery and validation of iridophore-specificalx4atranscription factor.

The cortico–basal ganglia–thalamo–cortical loop is one of the fundamental network motifs in the brain. Revealing its structural and functional organization is critical to understanding cognition, sensorimotor behaviour, and the natural history of many neurological and neuropsychiatric disorders. Classically, this network is conceptualized to contain three information channels: motor, limbic and associative1–4. Yet this three-channel view cannot explain the myriad functions of the basal ganglia. We previously subdivided the dorsal striatum into 29 functional domains on the basis of the topography of inputs from the entire cortex5. Here we map the multi-synaptic output pathways of these striatal domains through the globus pallidus external part (GPe), substantia nigra reticular part (SNr), thalamic nuclei and cortex. Accordingly, we identify 14 SNr and 36 GPe domains and a direct cortico-SNr projection. The striatonigral direct pathway displays a greater convergence of striatal inputs than the more parallel striatopallidal indirect pathway, although direct and indirect pathways originating from the same striatal domain ultimately converge onto the same postsynaptic SNr neurons. Following the SNr outputs, we delineate six domains in the parafascicular and ventromedial thalamic nuclei. Subsequently, we identify six parallel cortico–basal ganglia–thalamic subnetworks that sequentially transduce specific subsets of cortical information through every elemental node of the cortico– basal ganglia–thalamic loop. Thalamic domains relay this output back to the originating corticostriatal neurons of each subnetwork in a bona fide closed loop., Mesoscale connectomic mapping of the cortico–basal ganglia–thalamic network reveals key architectural and information processing features.

The role of natural selection in shaping biological diversity is an area of intense interest in modern biology. To date, studies of positive selection have primarily relied on genomic datasets from contemporary populations, which are susceptible to confounding factors associated with complex and often unknown aspects of population history. In particular, admixture between diverged populations can distort or hide prior selection events in modern genomes, though this process is not explicitly accounted for in most selection studies despite its apparent ubiquity in humans and other species. Through analyses of ancient and modern human genomes, we show that previously reported Holocene-era admixture has masked more than 50 historic hard sweeps in modern European genomes. Our results imply that this canonical mode of selection has probably been underappreciated in the evolutionary history of humans and suggest that our current understanding of the tempo and mode of selection in natural populations may be inaccurate. Ancient human genomes uncover historical hard sweeps Hard sweeps were common in ancient West Eurasian populations A historical hard sweep in the MHC-III region Admixture can obscure historical hard sweeps Evolutionary scenarios underlying sweep signal dilution The mutational basis of the Eurasian hard sweeps Discussion Methods

Meniscal injuries in elite athletes are a common cause of missed game time and even have the potential to be career shortening. In this patient group, care must be paid not only to the pathology, but also to a player’s contract status, time in the season, specific demands of his/her sport and position on the field, and future consequences. Successful treatment requires the clinician to understand the player’s goals and needs, communicate effectively between all stakeholders, and a have knowledge of the challenges posed by the different types of meniscal tear seen in this population. Paramount is the distinction between injuries to the medial and lateral meniscus. Deficiency of the lateral meniscus, as a result of a tear or a meniscectomy, leads to frequent early problems and inexorably to chondral degeneration thereby affecting an athlete’s ability to perform. Therefore, it is strongly recommended to repair the majority of lateral meniscal tears. Medial meniscal tears pose a more challenging treatment dilemma, as the success of partial meniscectomy in achieving reproducible, early return to play must be balanced against the long-term degenerative consequences. Many meniscal tears are correctly treated non-operatively. Level of evidence V.

Atlantic sturgeon (Acipenser oxyrinchus oxyrinchus) is a long-lived, anadromous species that is broadly distributed along the Atlantic coast of North America. Historic overharvest and habitat degradation resulted in significant declines to Atlantic sturgeon populations and, following decades of limited recovery, the species was listed under the Endangered Species Act of the United States in 2012. Given continued threats to recovery and limited information about population demography, there is a need for new tools to assist in Atlantic sturgeon conservation. Here, we present a range-wide microsatellite genetic baseline for North American Atlantic sturgeon that is comprised of 2510 individuals from 18 genetically distinct groups collected in 13 rivers and one estuary. Analysis of this baseline suggested that populations from the northern range of Atlantic sturgeon were more highly differentiated than those from the southern extent, where patterns of differentiation were complicated by rivers with genetically distinct spring and fall spawning runs and less geographic distance separating populations. Despite significant demographic bottleneck events, all populations showed at least moderate levels of genetic diversity across a suite of metrics. Additionally, individual-based assignment tests had over 80% accuracy for assigning individuals to their river of origin, highlighting the utility of this baseline for characterizing the composition of mixed-stock aggregations and understanding stock-specific vulnerability and recovery. The expanded spatial coverage of this baseline dataset enabled novel inferences about patterns of genetic differentiation and spawning phenology in Atlantic sturgeon which can be used to support conservation and management efforts.

Exhausted CD8 T cells (TEX) are a distinct state of T cell differentiation associated with failure to clear chronic viruses and cancer. Immunotherapies such as PD-1 blockade can reinvigorate TEX cells, but reinvigoration is not durable. A major unanswered question is whether TEX cells differentiate into functional durable memory T cells (TMEM) upon antigen clearance. Here, using a mouse model, we found that upon eliminating chronic antigenic stimulation, TEX cells partially (re)acquire phenotypic and transcriptional features of TMEM cells. These ‘recovering’ TEX cells originated from the T cell factor (TCF-1+) TEX progenitor subset. Nevertheless, the recall capacity of these recovering TEX cells remained compromised as compared to TMEM cells. Chromatin-accessibility profiling revealed a failure to recover core memory epigenetic circuits and maintenance of a largely exhausted open chromatin landscape. Thus, despite some phenotypic and transcriptional recovery upon antigen clearance, exhaustion leaves durable epigenetic scars constraining future immune responses. These results support epigenetic remodeling interventions for TEX cell–targeted immunotherapies. Wherry and colleagues examine whether exhausted T cells (TEX) can differentiate into functional memory T cells (TMEM) when chronic antigen is withdrawn. Using the chronic LCMV infection mouse model, they show that ‘recovering’ TEX cells (REC-TEX) only partially recover immunophenotypic and functional characteristics of TMEM cells. The epigenomic status of REC-TEX cells more closely resembles that of TEX cells, and, upon rechallenge, the REC-TEX cells were still compromised in their ability to respond to virus.

The rare, fatal neurodegenerative disorder Niemann-Pick disease type C1 (NPC1) arises from lysosomal accumulation of unesterified cholesterol and glycosphingolipids. These subcellular pathologies lead to phenotypes of hepatosplenomegaly, neurological degeneration and premature death. The timing and severity of NPC1 clinical presentation is extremely heterogeneous. This study analyzed RNA-Seq data from 42 NPC1 patient-derived, primary fibroblast cell lines to determine transcriptional changes induced by treatment with 2-hydroxypropyl-β-cyclodextrin (HPβCD), a compound currently under investigation in clinical trials. A total of 485 HPβCD-responsive genes were identified. Pathway enrichment analysis of these genes showed significant involvement in cholesterol and lipid biosynthesis. Furthermore, immunohistochemistry of the cerebellum as well as measurements of plasma from Npc1m1N null mice treated with HPβCD and adeno-associated virus gene therapy suggests that one of the identified genes, GPNMB, may serve as a useful biomarker of treatment response in NPC1 disease. Overall, this large NPC1 patient-derived dataset provides a comprehensive foundation for understanding the genomic response to HPβCD treatment.

A shotgun proteomics study using isobaric tags for relative and absolute quantification labeling was conducted to characterize proteins in chicken ovarian follicles immediately before and after cyclic recruitment. Granulosa cell (GC) layers from the most recently recruited follicle (GC9) and from each of the four largest prerecruitment follicles (GC1-4) plus theca tissue (TH) from the most recently recruited (TH9) and largest prerecruitment (TH1) follicles were compared. Of 1535 proteins identified, none were determined to be differentially expressed between TH9 and TH1. A pairwise comparison between GC9 and GC1, GC2, GC3, or GC4 resulted in one, five, five, and six differentially expressed proteins, respectively, including yolk and cholesterol transport proteins (vitellogenin 1-3 and apolipoprotein B). In addition, transforming growth factor-beta 1 and microRNA-21 pathways were predicted to be activated at recruitment. We also report, for the first time, the expression of the neuropeptide, RELAXIN-3 (RLN3), in GC. Quantitative polymerase chain reaction determined RLN3 expression to be highest in GC9 and GC1, but its receptors, RXFP1 and RXFP3, were highest in TH and ovarian stroma, respectively. Overall, cyclic recruitment is associated with changes in protein expression predominantly within follicle GC, and a potential role for RLN3 in follicle recruitment and the initiation of GC differentiation warrants further investigation.

The objective of the study was to determine if psychological readiness for sport and knee self-efficacy assessed early (3 months) after anterior cruciate ligament reconstruction (ACLR) are predictive of self-reported functional outcomes, quadriceps strength, and knee mechanics while running at the time of return to sport training (6 months). Thirty athletes with unilateral ACLR completed the ACL Return to Sport after Injury (ACL-RSI) and Knee Self-Efficacy Scale (K-SES) 3 months after ACLR and completed self-reported functional outcomes, isometric quadriceps strength testing, and three-dimensional running gait analysis 6 months after ACLR. The 3-month ACL-RSI significantly correlated with the 6-month International Knee Documentation Committee (IKDC; r = 0.565, p = 0.001), Knee Injury and Osteoarthritis Outcome Score (KOOS) sport/recreational activities (KOOSSport ; r = 0.548, p = 0.002) and quality of life (KOOSQoL ; r = 0.431, p = 0.017), and quadriceps strength (r = 0.528, p = 0.003). The 3-month K-SES significantly correlated with the 6-month IKDC (r = 0.528, p = 0.003), KOOSSport (r = 0.430, p = 0.018), KOOSQoL (r = 0.411, p = 0.024), quadriceps strength (r = 0.465, p = 0.010), and knee flexion excursion (r = 0.472, p = 0.008). With multivariate modeling, both the ACL-RSI and K-SES were predictive of the IKDC (R2 = 0.411; p = 0.001). Only the ACL-RSI was predictive of the KOOSSport (R2 = 0.300; p = 0.002), KOOSQoL (R2 = 0.186; p = 0.017), and quadriceps strength (R2 = 0.279; p = 0.003), whereas only the K-SES was predictive of knee flexion excursion (R2 = 0.173; p = 0.022). Athletes with greater psychological readiness for sport and knee self-efficacy at 3 months demonstrated higher scores on self-reported functional outcomes, greater quadriceps strength, and greater knee flexion excursion at 6 months after ACLR. This study indicates that psychosocial measures may be important to include into early post-surgical evaluations to help guide and facilitate interventions to restore subjective and objective knee function.

Despite the important function of neutrophils in the eradication of infections and induction of inflammation, the molecular mechanisms regulating the activation and termination of the neutrophil immune response is not well understood. Here, the function of the small GTPase from the RGK family, Gem, is characterized as a negative regulator of the NADPH oxidase through autophagy regulation. Gem knockout (Gem KO) neutrophils show increased NADPH oxidase activation and increased production of extracellular and intracellular reactive oxygen species (ROS). Enhanced ROS production in Gem KO neutrophils was associated with increased NADPH oxidase complex-assembly as determined by quantitative super-resolution microscopy, but normal exocytosis of gelatinase and azurophilic granules. Gem-deficiency was associated with increased basal autophagosomes and autolysosome numbers but decreased autophagic flux under phorbol ester-induced conditions. Neutrophil stimulation triggered the localization of the NADPH oxidase subunits p22phox and p47phox at LC3-positive structures suggesting that the assembled NADPH oxidase complex is recruited to autophagosomes, which was significantly increased in Gem KO neutrophils. Prevention of new autophagosome formation by treatment with SAR405 increased ROS production while induction of autophagy by Torin-1 decreased ROS production in Gem KO neutrophils, and also in wild-type neutrophils, suggesting that macroautophagy contributes to the termination of NADPH oxidase activity. Autophagy inhibition decreased NETs formation independently of enhanced ROS production. NETs production, which was significantly increased in Gem-deficient neutrophils, was decreased by inhibition of both autophagy and calmodulin, a known GEM interactor. Intracellular ROS production was increased in Gem KO neutrophils challenged with live Gram-negative bacteria Pseudomonas aeruginosa or Salmonella Typhimurium, but phagocytosis was not affected in Gem-deficient cells. In vivo analysis in a model of Salmonella Typhimurium infection indicates that Gem-deficiency provides a genetic advantage manifested as a moderate increased in survival to infections. Altogether, the data suggest that Gem-deficiency leads to the enhancement of the neutrophil innate immune response by increasing NADPH oxidase assembly and NETs production and that macroautophagy differentially regulates ROS and NETs in neutrophils.

Rationale CC16 (club cell secretory protein) is a pneumoprotein produced predominantly by pulmonary club cells. Circulating CC16 is associated with protection from the inception and progression of ...

Objectives This study aimed to characterize trends in technetium Tc 99m pyrophosphate (99mTc-PYP) scanning for amyloid transthyretin cardiac amyloidosis (ATTR-CA) diagnosis, to determine whether patients underwent appropriate assessment with monoclonal protein and genetic testing, to evaluate use of single-photon emission computed tomography (SPECT) in addition to planar imaging, and to identify predictive factors for ATTR-CA. Background 99mTc-PYP scintigraphy has been repurposed for noninvasive diagnosis of ATTR-CA. Increasing use of 99mTc-PYP can facilitate identification of ATTR-CA, but appropriate use is critical for accurate diagnosis in an era of high-cost targeted therapeutics. Methods Patients undergoing 99mTc-PYP scanning 1 h after injection at a quaternary care center from 2010 to 2019 were analyzed; clinical information was abstracted; and SPECT results were analyzed. Results Over the decade, endomyocardial biopsy rates remained stable with scanning rates peaking at 132 in 2019 (p Conclusions Use of noninvasive testing with 99mTc-PYP scanning for evaluation of ATTR-CA is increasing, and the inclusion of monoclonal protein testing and SPECT imaging is crucial to rule out amyloid light chain amyloidosis and distinguish myocardial retention from blood pooling.

Posterior knee pain after total knee arthroplasty (TKA) is common despite multimodal analgesia and regional anesthesia use. This review included randomized controlled trials (RCTs) comparing analgesic outcomes after inclusion of local anesthetic infiltration between the popliteal artery and capsule of the knee (iPACK) block versus pathways without iPACK. Electronic databases (MEDLINE, Cochrane Library, Web of Science, Scopus) were searched from inception to 10/11/2020. Eligible studies evaluated iPACK use on primary outcomes: opioid consumption and pain scores with movement. Secondary outcomes included rest pain, patient satisfaction, length of stay (LOS), gait distance, knee range of motion (ROM), and complications. Bias and quality were appraised using the Cochrane Risk of Bias tool and Grading of Recommendations, Assessment, Development, and Evaluations (GRADE) guidelines. Eight RCTs (777 patients) were included. iPACK block use demonstrated similar opioid consumption in the PACU (4/7 RCTs) and 24 hours after TKA (5/7 RCTs) compared to without iPACK (moderate-quality GRADE evidence). Additionally, iPACK block use demonstrated lower movement pain scores in PACU (3/5 RCTs) but similar or higher pain scores after 24 hours (5/7 RCTs; low-quality GRADE evidence). Studies consistently reported no difference in gait distance (4/4 RCTs) or complications (7/7 RCTs) between treatment arms (high-quality GRADE evidence), although differing effect estimates were observed with resting pain, satisfaction, LOS, and knee ROM. This review provides a foundation of knowledge on iPACK efficacy. While evidence does not currently support widespread inclusion of iPACK within enhanced recovery pathways for TKA, limitations suggest further study is warranted.

Subcutaneous injection site reactions to sustained-release buprenorphine hydrochloride (Buprenorphine SR) in macaques have been reported in only a single case report. In the current study, we evaluated the incidence rate and predictors of buprenorphine SR reactions in the subcutaneous tissue of rhesus macaques (Macaca mulatta) based on retrospective review of macaque buprenorphine SR injection records. Potentially predictive variables were identified with logistic regression modeling and were evaluated using model selection based on Akaike information criterion. Record review revealed sub- cutaneous tissue reactions occurred in 52 (3%) of 1559 injections and were noted between 4 and 311 d after injection. Model selection showed that body weight and MHC allele Mamu-B*29 were the best predictors of subcutaneous reactions. Based on these results, we recommend consideration of potential risk factors prior to the administration of buprenorphine SR to a rhesus macaque. In addition, the authors advise that using the highest concentration of buprenorphine SR available may reduce injection site reaction rates due to the injection of less copolymer.

Atlantic sturgeon (Acipenser oxyrinchus oxyrinchus) is a large, anadromous fish native to the Atlantic Coast of North America. Although this species once supported important fisheries, centuries of exploitation and habitat degradation have resulted in dramatic declines, presumed extirpation in some rivers, and ultimately listing under the U.S. Endangered Species Act (ESA). Under the ESA, Atlantic sturgeon are listed as five separate Distinct Population Segments (DPSs), which form the basis for federal management. Despite state and federal protections Atlantic sturgeon still face significant threats to their recovery, including fisheries bycatch mortality, marine construction, dredging, dams, and vessel strikes. However, because subadult and adult Atlantic sturgeon migrate extensively across estuarine and marine environments and frequently form mixed-stock aggregations in non-natal habitats, it can be difficult to determine how these threats impact specific populations and DPSs. To better understand ontogenetic shifts in habitat use and stock-specific exposure to anthropogenic threats, we performed a mixed-stock analysis of 1704 Atlantic sturgeon encountered across the U.S. Atlantic Coast. Collections made north of Cape Cod, MA and south of Cape Hatteras, NC were dominated by individuals from regional stocks; however, we found extensive stock mixing in the mid-Atlantic region, particularly in coastal environments where individuals from all five DPSs were commonly observed. Subadults and adults that were encountered in offshore environments had moved, on average, 277 km from their natal source; however, 23% were sampled over 500 km from their natal river suggesting long-distance movements are relatively common in these age classes. Overall, our work highlights that Atlantic sturgeon populations are vulnerable to threats over vast areas and emphasizes the need for continued genetic monitoring to track recovery progress.

BackgroundGastric adenocarcinoma (GAC) is a lethal disease with limited therapeutic options. Genetic alterations in chromatin remodelling gene AT-rich interactive domain 1A (ARID1A) and mTOR pathway activation occur frequently in GAC. Targeting the mechanistic target of rapamycin (mTOR) pathway in unselected patients has failed to show survival benefit. A deeper understanding of GAC might identify a subset that can benefit from mTOR inhibition.MethodsGenomic alterations in ARID1A were analysed in GAC. Mouse gastric epithelial cells from CK19-Cre-Arid1Afl/fl and wild-type mice were used to determine the activation of oncogenic genes due to loss of Arid1A. Functional studies were performed to determine the significance of loss of ARID1A and the sensitivity of ARID1A-deficient cancer cells to mTOR inhibition in GAC.ResultsMore than 30% of GAC cases had alterations (mutations or deletions) of ARID1A and ARID1A expression was negatively associated with phosphorylation of S6 and SOX9 in GAC tissues and patient-derived xenografts (PDXs). Activation of mTOR signalling (increased pS6) and SOX9 nuclear expression were strongly increased in Arid1A−/− mouse gastric tissues which could be curtailed by RAD001, an mTOR inhibitor. Knockdown of ARID1A in GAC cell lines increased pS6 and nuclear SOX9 and increased sensitivity to an mTOR inhibitor which was further amplified by its combination with fluorouracil both in vitro and in vivo in PDXs.ConclusionsThe loss of ARID1A activates pS6 and SOX9 in GAC, which can be effectively targeted by an mTOR inhibitor. Therefore, our studies suggest a new therapeutic strategy of clinically targeting the mTOR pathway in patients with GAC with ARID1A deficiency.

Background Flowers which imitate insect oviposition sites probably represent the most widespread form of floral mimicry, exhibit the most diverse floral signals and are visited by two of the most speciose and advanced taxa of insect – beetles and flies. Detailed comparative studies on brood-site mimics pollinated exclusively by each of these insect orders are lacking, limiting our understanding of floral trait adaptation to different pollinator groups in these deceptive systems. Methods Two closely related and apparent brood-site mimics, Typhonium angustilobum and T. wilbertii (Araceae) observed to trap these distinct beetle and fly pollinator groups were used to investigate potential divergence in floral signals and traits most likely to occur under pollinator-mediated selection. Trapped pollinators were identified and their relative abundances enumerated, and thermogenic, visual and chemical signals and morphological traits were examined using thermocouples and quantitative reverse transcription–PCR, reflectance, gas chromatography–mass spectrometry, floral measurements and microscopy. Key Results Typhonium angustilobum and T. wilbertii were functionally specialized to trap saprophagous Coleoptera and Diptera, respectively. Both species shared similar colour and thermogenic traits, and contained two highly homologous AOX genes (AOX1a and AOX1b) most expressed in the thermogenic tissue and stage (unlike pUCP). Scent during the pistillate stage differed markedly – T. angustilobum emitted a complex blend of sesquiterpenes, and T. wilbertii, a dung mimic, emitted high relative amounts of skatole, p-cresol and irregular terpenes. The species differed significantly in floral morphology related to trapping mechanisms. Conclusions Functional specialization and pollinator divergence were not associated with differences in anthesis rhythm and floral thermogenic or visual signals between species, but with significant differences in floral scent and morphological features, suggesting that these floral traits are critical for the attraction and filtering of beetle or fly pollinators in these two brood-site mimics.

Key points The aim was to determine whether detachment of the tectorial membrane (TM) from the organ of Corti in Tecta/Tectb-/- mice affects the biophysical properties of cochlear outer hair cells (OHCs). Tecta/Tectb-/- mice have highly elevated hearing thresholds, but OHCs mature normally. Mechanoelectrical transducer (MET) channel resting open probability (Po ) in mature OHC is ∼50% in endolymphatic [Ca2+ ], resulting in a large standing depolarizing MET current that would allow OHCs to act optimally as electromotile cochlear amplifiers. MET channel resting Po in vivo is also high in Tecta/Tectb-/- mice, indicating that the TM is unlikely to statically bias the hair bundles of OHCs. Distortion product otoacoustic emissions (DPOAEs), a readout of active, MET-dependent, non-linear cochlear amplification in OHCs, fail to exhibit long-lasting adaptation to repetitive stimulation in Tecta/Tectb-/- mice. We conclude that during prolonged, sound-induced stimulation of the cochlea the TM may determine the extracellular Ca2+ concentration near the OHC's MET channels. Abstract The tectorial membrane (TM) is an acellular structure of the cochlea that is attached to the stereociliary bundles of the outer hair cells (OHCs), electromotile cells that amplify motion of the cochlear partition and sharpen its frequency selectivity. Although the TM is essential for hearing, its role is still not fully understood. In Tecta/Tectb-/- double knockout mice, in which the TM is not coupled to the OHC stereocilia, hearing sensitivity is considerably reduced compared with that of wild-type animals. In vivo, the OHC receptor potentials, assessed using cochlear microphonics, are symmetrical in both wild-type and Tecta/Tectb-/- mice, indicating that the TM does not bias the hair bundle resting position. The functional maturation of hair cells is also unaffected in Tecta/Tectb-/- mice, and the resting open probability of the mechanoelectrical transducer (MET) channel reaches values of ∼50% when the hair bundles of mature OHCs are bathed in an endolymphatic-like Ca2+ concentration (40 μM) in vitro. The resultant large MET current depolarizes OHCs to near -40 mV, a value that would allow optimal activation of the motor protein prestin and normal cochlear amplification. Although the set point of the OHC receptor potential transfer function in vivo may therefore be determined primarily by endolymphatic Ca2+ concentration, repetitive acoustic stimulation fails to produce adaptation of MET-dependent otoacoustic emissions in vivo in the Tecta/Tectb-/- mice. Therefore, the TM is likely to contribute to the regulation of Ca2+ levels around the stereocilia, and thus adaptation of the OHC MET channel during prolonged sound stimulation.

Recent studies have evaluated alternatives to the use of live animals in colony health monitoring. Currently, an alternative method that is suitable for all rack types and that has been verified to detect the infectious agents most commonly excluded from mouse colonies is unavailable. We compared the use of filter paper placed on the inside floor of mouse cages to the traditional use of sentinel mice in the detection of several prevalent murine pathogens including mouse hepatitis virus (MHV), murine norovirus (MNV), minute virus of mice (MVM), mouse parvovirus (MPV), Theiler murine encephalomyelitis virus (TMEV), Helicobacter spp., Syphacia obvelata, and Aspiculuris tetraptera. Experimental groups comprised 7 cages containing either 2 pieces of filter paper on the cage floor or 2 ICR sentinel mice. Soiled bedding from pet-store mice was transferred to the experimental cages weekly for 8 wk. At 1 and 2 mo after bedding transfer, the filter papers were evaluated by PCR and sentinel mice were tested by serology and fecal PCR. Filter papers detected all pathogens as effectively (MHV, MNV, MPV, MVM, TMEV S. obvelata, and A. tetraptera) or more effectively (Helicobacter spp.) than sentinel mice at both time points. Filter papers more readily detected pathogens with a high copy number per RT-PCR analysis than a low copy number. Helicobacter spp. were not detected by sentinel mice at either time point. These results indicate that the use of filter paper placed on the interior floor of empty mouse cages and exposed to soiled bedding is efficient in detecting bacteria, endoparasites, and most of the common mouse viruses included in an animal health monitoring program.

Sesquitepenoids inuloxins A-D, belonging to different subgroups, were isolated from Dittrichia viscosa and showed potential biocontrol of some parasitic plants as Pelipanche, Orobanche, and Cuscuta species. The absolute configurations of the first three inuloxins A-C were previously determined by using experimental and computational chiroptical spectroscopic methods. The absolute configuration of inuloxin D remains to be established. The bioactive inuloxin E, closely related to inuloxin D, was recently isolated from the same plant organic extract. The same relative configuration of inuloxin D was assigned to inuloxin E by comparison of their NMR spectroscopic data. The absolute configurations of inuloxin D and inuloxin E are suggested in this work by analysis of the experimental and predicted chiroptical properties of the 4-O-acetyl derivative of inuloxin D.

Background Studies of the gut microbiome are becoming increasingly important. Such studies require stool collections that can be processed or frozen in a timely manner so as not to alter the microbial content. Due to the logistical difficulties of home-based stool collection, there has been a challenge in selecting the appropriate sample collection technique and comparing results from different microbiome studies. Thus, we compared stool collection and two alternative clinic-based fecal microbiome collection techniques, including a newer glove-based collection method. Results We prospectively enrolled 22 adult men from our prostate cancer screening cohort SABOR (San Antonio Biomarkers of Risk for prostate cancer) in San Antonio, TX, from 8/2018 to 4/2019. A rectal swab and glove tip sample were collected from each participant during a one-time visit to our clinics. A single stool sample was collected at the participant’s home. DNA was isolated from the fecal material and 16 s rRNA sequencing of the V1-V2 and V3-V4 regions was performed. We found the gut microbiome to be similar in richness and evenness, noting no differences in alpha diversity among the collection methods. The stool collection method, which remains the gold-standard method for the gut microbiome, proved to have different community composition compared to swab and glove tip techniques (pp> 0.05). Despite differences between home-based stool and office-based fecal collection methods, we noted that the distance metrics for the three methods cluster by participant indicating within-person similarities. Additionally, no taxa differed among the methods in a Linear Discriminant Analysis Effect Size (LEfSe) analysis comparing all-against-all sampling methods. Conclusion The glove tip method provides similar gut microbiome results as rectal swab and stool microbiome collection techniques. The addition of a new office-based collection technique could help easy and practical implementation of gut microbiome research studies and clinical practice.

Globally, one in nine people suffer from undernourishment with evidence that this number is increasing. Additionally, due to the projected 50% increase in global population, the demand in worldwide animal-sourced protein is expected to double by 2050. Furthermore, not only are global animal protein supply chains susceptible to the effects of climate change, but they are also a significant contributor to greenhouse gas emissions and require large areas of arable land. Single cell proteins (SCPs) are an alternative protein source that offer a potential route to reduce the environmental impact of global protein consumption. One such SCP is Fusarium venenatum, which is a strain of mycoprotein widely sold under the brand name Quorn and is produced through the fermentation of the microorganism on glucose. However, this glucose still has a significant arable land-use burden associated with it. In order to mitigate this burden, sugars derived from agricultural lignocellulosic residues could be used, however, additional processing steps are required. In this work, exploratory research on fermentation of F. venenatum on sugars derived from lignocellulosic residues is presented. The food-grade ionic liquid [Ch][HSO4] was employed in combination with food-grade Celluclast 1.5L to extract glucose from rice straw residues, which resulted in an overall glucose yield of 42.4% compared to using non-food certified ionic liquid [TEA][HSO4], which yielded 92.8%. Based on these results, a Techno-Economic Analysis (TEA) and Life Cycle Assessment (LCA) were conducted after synthesising a biorefinery process model. TEA modelling outcomes highlighted that the crude mycoprotein paste product could be produced for $5.04 kg−1 ($40.04 per kg-protein). By conducting a retro-techno-economic analysis, it was shown that there is reasonable scope for reducing this price further by improving saccharification yields and utilising feedstocks with high cellulose contents. Furthermore, LCA results demonstrated significant sustainability benefits of lignocellulosic-derived mycoprotein, with greenhouse gas emissions less than 14% that of protein from beef. However, the most significant advantage of this technology is the minimal dependence on arable land compared to animal-sourced and plant-sourced proteins such as beef and tofu.

Undergraduate students participating in the UCLA Undergraduate Research Consortium for Functional Genomics (URCFG) have conducted a two-phased screen using RNA interference (RNAi) in combination with fluorescent reporter proteins to identify genes important for hematopoiesis in Drosophila. This screen disrupted the function of approximately 3500 genes and identified 137 candidate genes for which loss of function leads to observable changes in the hematopoietic development. Targeting RNAi to maturing, progenitor, and regulatory cell types identified key subsets that either limit or promote blood cell maturation. Bioinformatic analysis reveals gene enrichment in several previously uncharacterized areas, including RNA processing and export and vesicular trafficking. Lastly, the participation of students in this course-based undergraduate research experience (CURE) correlated with increased learning gains across several areas, as well as increased STEM retention, indicating that authentic, student-driven research in the form of a CURE represents an impactful and enriching pedagogical approach.

We examined the changes in mechanical strain response of male and female mouse tibia and ulna, using axial compression tests, to assess age-related changes in tibiae and ulnae by a non-contact strain measurement technique called the digital image correlation (DIC) and the standard strain gage. A unique aspect of the study was to compare bones from the same animal to study variations in behavior with aging. This study was conducted using male and female C57Bl/6 mice at 6, 12 and 22 months of age (N=6-7 per age and sex) using three load levels. The DIC technique was able to detect a greater number of statistically significant differences in comparison to the strain gaging method. Male ulna showed significantly higher DIC strains compared to strains captured from strain gage at all three levels of load at 6 months and in the lowest load at 12 months. DIC measurements revealed that the ulna becomes stiffer with aging for both males and females, which resulted in 0.4 to 0.8 times reduced strains in the 22-month group compared to the 6 month group. Male tibia showed three-fold increased strains in the 22 months group at 11.5 N load compared to 6 months group (p

Cryptococcus gattii was diagnosed in two female indoor-housed rhesus macaques. Gross and histopathologic findings included an isolated pulmonary cryptococcoma in a non-SIV-infected macaque and disseminated disease centered on the lungs of an SIV-infected macaque. Fungal yeast were positive with special stains, and the diagnoses were confirmed with a lateral flow assay and PCR.

Compared with our closest living evolutionary cousins, humans appear unusually prone to develop carcinomas (cancers arising from epithelia). The SIGLEC12 gene, which encodes the Siglec‐XII protein expressed on epithelial cells, has several uniquely human features: a fixed homozygous missense mutation inactivating its natural ligand recognition property; a polymorphic frameshift mutation eliminating full‐length protein expression in ~60%–70% of worldwide human populations; and, genomic features suggesting a negative selective sweep favoring the pseudogene state. Despite the loss of canonical sialic acid binding, Siglec‐XII still recruits Shp2 and accelerates tumor growth in a mouse model. We hypothesized that dysfunctional Siglec‐XII facilitates human carcinoma progression, correlating with known tumorigenic signatures of Shp2‐dependent cancers. Immunohistochemistry was used to detect Siglec‐XII expression on tissue microarrays. PC‐3 prostate cancer cells were transfected with Siglec‐XII and transcription of genes enriched with Siglec‐XII was determined. Genomic SIGLEC12 status was determined for four different cancer cohorts. Finally, a dot blot analysis of human urinary epithelial cells was established to determine the Siglec‐XII expressors versus non‐expressors. Forced expression in a SIGLEC12 null carcinoma cell line enriched transcription of genes associated with cancer progression. While Siglec‐XII was detected as expected in ~30%–40% of normal epithelia, ~80% of advanced carcinomas showed strong expression. Notably, >80% of late‐stage colorectal cancers had a functional SIGLEC12 allele, correlating with overall increased mortality. Thus, advanced carcinomas are much more likely to occur in individuals whose genomes have an intact SIGLEC12 gene, likely because the encoded Siglec‐XII protein recruits Shp2‐related oncogenic pathways. The finding has prognostic, diagnostic, and therapeutic implications.

Natural hail can cause significant damage on seed yield and yield contributing traits of canola (Brassica napus L.). Hail damage can be assessed by (i) type of damage such as stand reduction, stem cut-off, and leaf defoliation, (ii) level of damage, and (iii) plant growth stage. In this research, a simulated hail study was performed by applying nonuniform stand reduction treatments on canola grown in North Dakota, USA, over 5 site-years, in 2017 and 2018. The experiment was a randomized complete block design 4 × 5 factorial arrangement with four growth stages, rosette, bolting, 50%, and 90% flowering, when five stand reduction treatments were applied at 0 (control), 25%, 50%, 75%, and 90%. Growth stage and stand reduction were significant for seed yield where yield decreased as stage of treatment progressed and level of stand reduction increased. Regression equations were developed to estimate the seed yield reduction at each growth stage as stand reduction increased. Stand reduction also affected other traits where plant height was reduced as stand reduction increased, whereas 1000-seed weight, primary branches plant−1, secondary branches plant−1, pods plant−1, seed yield plant−1, plant biomass plant−1, and harvest index plant−1 increased as stand reduction increased. As growth stage progressed the number of primary branches plant−1, secondary branches plant−1, pods plant−1, and harvest index plant−1 decreased, whereas 1000-seed weight increased. The findings of differential yield losses by stand reduction will help producers and crop adjusters to assess the severity of hail damage in canola.

Species of the genus Euphausia dominate the euphausiid biomass of the Southern Ocean, the three largest being Euphausia superba, E. triacantha and E. crystallorophias. We measured a number of morphological features to identify differences between, and within, these species to obtain ecological insights. Interspecifically, the greatest difference was carapace size, with that of E. superba being by far the largest and most variable. This likely reflects its prolific spawning capacity compared with other euphausiid species. E. triacantha exhibited an extended sixth abdominal segment that could facilitate greater levels of thrust in the tail flip escape response. The pleopods, which provide propulsion in forward swimming, were more than 50% larger in E. superba, indicating a greater capacity for directional movement at high velocities. E. crystallorophias had eyes that were almost double the size of those in E. superba and E. triacantha, which may help retain visual resolution within its under-ice habitat. Intraspecifically, we found the above morphological features differed little between sexes and developmental stages in E. crystallorophias and E. triacantha, but differed significantly in E. superba. Compared to females and juveniles, male E. superba had significantly larger eyes and pleopods, whilst the carapace in males became shorter as a proportion of body length during growth. These features indicate a greater capacity for searching and swimming in males, which, we hypothesise, increases their ability to locate and fertilise females. This morphological specialisation in male E. superba is indicative of comparatively greater inter-male competition resulting from its tendency to form large, dense swarms.

Triple-negative breast cancers contain a spectrum of epithelial and mesenchymal phenotypes. SUM-229PE cells represent a model for this heterogeneity, maintaining both epithelial and mesenchymal subpopulations that are genomically similar but distinct in gene expression profiles. We identified differential regions of open chromatin in epithelial and mesenchymal cells that were strongly correlated with regions of H3K27ac. Motif analysis of these regions identified consensus sequences for transcription factors that regulate cell identity. Treatment with the MEK inhibitor trametinib induced enhancer remodeling that is associated with transcriptional regulation of genes in epithelial and mesenchymal cells. Motif analysis of enhancer peaks downregulated in response to chronic treatment with trametinib identified AP-1 motif enrichment in both epithelial and mesenchymal subpopulations. Chromatin immunoprecipitation sequencing (ChIP-seq) of JUNB identified subpopulation-specific localization, which was significantly enriched at regions of open chromatin. These results indicate that cell identity controls localization of transcription factors and chromatin-modifying enzymes to enhancers for differential control of gene expression. We identified increased H3K27ac at an enhancer region proximal to CXCR7, a G-protein–coupled receptor that increased 15-fold in expression in the epithelial subpopulation during chronic treatment. RNAi knockdown of CXCR7 inhibited proliferation in trametinib-resistant cells. Thus, adaptive resistance to chronic trametinib treatment contributes to proliferation in the presence of the drug. Acquired amplification of KRAS following trametinib dose escalation further contributed to POS cell proliferation. Adaptive followed by acquired gene expression changes contributed to proliferation in trametinib-resistant cells, suggesting inhibition of early transcriptional reprogramming could prevent resistance and the bypass of targeted therapy. Implications: We defined the differential responses to trametinib in subpopulations of a clinically relevant in vitro model of TNBC, and identified both adaptive and acquired elements that contribute to the emergence of drug resistance mediated by increased expression of CXCR7 and amplification of KRAS.

Loss of the tumor suppressor NF1 leads to activation of RAS effector pathways, which are therapeutically targeted by inhibition of mTOR (mTORi) or MEK (MEKi). However, therapeutic inhibition of RAS effectors leads to the development of drug resistance and ultimately disease progression. To investigate molecular signatures in the context of NF1 loss and subsequent acquired drug resistance, we analyzed the exomes, transcriptomes, and kinomes of Nf1-mutant mouse tumor cell lines and derivatives of these lines that acquired resistance to either MEKi or mTORi. Biochemical comparisons of this unique panel of tumor cells, all of which arose in Nf1+/− mice, indicate that loss of heterozygosity of Nf1 as an initial genetic event does not confer a common biochemical signature or response to kinase inhibition. Although acquired drug resistance by Nf1-mutant tumor cells was accompanied by altered kinomes and irreversibly altered transcriptomes, functionally in multiple Nf1-mutant tumor cell lines, MEKi resistance was a stable phenotype, in contrast to mTORi resistance, which was reversible. Collectively, these findings demonstrate that Nf1-mutant tumors represent a heterogeneous group biochemically and undergo broader remodeling of kinome activity and gene expression in response to targeted kinase inhibition.

Sperm velocity is a key trait that predicts the outcome of sperm competition. By promoting or impeding sperm velocity, females can control fertilization via postcopulatory cryptic female choice. In Chinook salmon, ovarian fluid (OF), which surrounds the ova, mediates sperm velocity according to male and female identity, biasing the outcome of sperm competition towards males with faster sperm. Past investigations have revealed proteome variation in OF, but the specific components of OF that differentially mediate sperm velocity have yet to be characterized. Here we use quantitative proteomics to investigate whether OF protein composition explains variation in sperm velocity and fertilization success. We found that OF proteomes from six females robustly clustered into two groups and that these groups are distinguished by the abundance of a restricted set of proteins significantly associated with sperm velocity. Exposure of sperm to OF from females in group I had faster sperm compared to sperm exposed to the OF of group II females. Overall, OF proteins that distinguished between these groups were enriched for vitellogenin and calcium ion interactions. Our findings suggest that these proteins may form the functional basis for cryptic female choice via the biochemical and physiological mediation of sperm velocity.

Background Serratia marcescens is a well-known cause of nosocomial infectious outbreaks in the neonatal intensive care unit, with a high mortality rate in the vulnerable preterm population. However, it is not typically associated with neonatal sepsis secondary to intrapartum vertical transmission. We present the case of a preterm male born at 25 weeks and 4 days of gestation in Okinawa, Japan with culture-proven S. marcescens chorioamnionitis and sepsis, as well as a review of the previously published literature. Methods We conducted a literature search utilizing MeSH indexing with the headings [chorioamnionitis], [Serratia], and [infant, newborn] limited to "humans" with a publication date range between 1950 and 2020. Results All reported cases of preterm S. marcescens chorioamnionitis occurred in coastal locations. The majority of cases resulted in spontaneous abortion, and we found no published reports of confirmed S. marcescens chorioamnionitis in conjunction with viable preterm delivery and positive neonatal cultures. In the case presented herein, S. marcescens chorioamnionitis with associated neonatal sepsis was confirmed by positive placental and blood cultures. Bacterial clearance was achieved following an antibiotic course consisting of 5 days of gentamicin and 14 days of meropenem therapy. Conclusions S. marcescens is an uncommon cause of chorioamnionitis that can have devastating neonatal consequences, especially in the at-risk preterm population.

Bayesian stable isotope mixing models (BSIMMs) for δ13 C and δ15 N can be a useful tool to reconstruct diets, characterize trophic relationships, and assess spatiotemporal variation in food webs. However, use of this approach typically requires a priori knowledge on the level of enrichment occurring between the diet and tissue of the consumer being sampled (i.e. a trophic discrimination factor or TDF). Trophic discrimination factors derived from captive feeding studies are highly variable, and it is challenging to select the appropriate TDF for diet estimation in wild populations. We introduce a novel method for estimating TDFs in a wild population-a proportionally balanced equation that uses high-precision diet estimates from nest cameras installed on a subset of nests in lieu of a controlled feeding study (TDFCAM ). We tested the ability of BSIMMs to characterize diet in a free-living population of gyrfalcon Falco rusticolus nestlings by comparing model output to high-precision nest camera diet estimates. We analysed the performance of models formulated with a TDFCAM against other relevant TDFs and assessed model sensitivity to an informative prior. We applied the most parsimonious model inputs to a larger sample to analyse broad-scale temporal dietary trends. Bayesian stable isotope mixing models fitted with a TDFCAM and uninformative prior had the best agreement with nest camera data, outperforming TDFs derived from captive feeding studies. BSIMMs produced with a TDFCAM produced reliable diet estimates at the nest level and accurately identified significant temporal shifts in gyrfalcon diet within and between years. Our method of TDF estimation produced more accurate estimates of TDFs in a wild population than traditional approaches, consequently improving BSIMM diet estimates. We demonstrate how BSIMMs can complement a high-precision diet study by expanding its spatiotemporal scope of inference and recommend this integrative methodology as a powerful tool for future trophic studies.

The Hsp90 molecular chaperone is required for the function of hundreds of different cellular proteins. Hsp90 and a cohort of interacting proteins called cochaperones interact with clients in an ATP-dependent cycle. Cochaperone functions include targeting clients to Hsp90, regulating Hsp90 ATPase activity, and/or promoting Hsp90 conformational changes as it progresses through the cycle. Over the last 20 years, the list of cochaperones identified in human cells has grown from the initial six identified in complex with steroid hormone receptors and protein kinases to about fifty different cochaperones found in Hsp90-client complexes. These cochaperones may be placed into three groups based on shared Hsp90 interaction domains. Available evidence indicates that cochaperones vary in client specificity, abundance, and tissue distribution. Many of the cochaperones have critical roles in regulation of cancer and neurodegeneration. A more limited set of cochaperones have cellular functions that may be limited to tissues such as muscle and testis. It is likely that a small set of cochaperones are part of the core Hsp90 machinery required for the folding of a wide range of clients. The presence of more selective cochaperones may allow greater control of Hsp90 activities across different tissues or during development. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12192-020-01167-0) contains supplementary material, which is available to authorized users.

Background Squamous cell carcinoma is a commonly reported neoplasm in avian species. Often locally invasive, it rarely distantly metastasizes. Superficial chronic ulcerative dermatitis (SCUD) is a commonly observed dermatologic condition in companion psittacine birds. Case description This report describes a case of metastatic squamous cell carcinoma in an African gray parrot (Psittacus erithacus) caused by malignant transformation of an initial SCUD lesion that developed over a 2.5-year period. The right humerus was previously amputated to treat a squamous cell carcinoma, but it recurred in the skin 4 months prior to presentation. The bird presented on referral with pelvic limb paresis, and computed tomography showed a large, more right sided, paraspinal mass involving the right caudal coelom and effacing the lungs, ribs, and vertebral canal, resulting in the presenting neurologic signs. The patient was euthanized, and limited necropsy diagnosed the coelomic mass as a squamous cell carcinoma effacing the liver. Conclusions Though uncommonly reported, squamous cell carcinoma may distantly metastasize to other parts of the body even with partial or seemingly complete excision. SCUD is still a poorly understood condition and should be further researched to determine etiology and find more effective therapies. Case relevance This is the first report of a metastatic squamous cell carcinoma invading the vertebral canal and spinal cord resulting in upper motor neuron signs in the pelvic limbs. Chronic inflammation may lead to malignant transformation, and once squamous cell carcinoma has developed, the goal of therapy should be complete surgical excision where possible.

Background The GENE-UP® Listeria spp. 2 (LIS 2) assay (Performance Tested MethodSM 121803) is a real-time PCR molecular detection method for the rapid detection of Listeria species (Listeria monocytogenes, L. innocua, L. ivanovii, L. seeligeri, and L. welshimeri) in a variety of foods and environmental surfaces. Objective The purpose of this validation was to evaluate the method’s interlaboratory performance and submit the results to AOAC INTERNATIONAL for adoption as First Action Official MethodSM for the detection of Listeria species in a variety of foods and select environmental surfaces. Method The GENE-UP method was evaluated in a multi-laboratory study as part of the AFNOR NF VALIDATION certification process using unpaired test portions for one food matrix, full-cream goat milk cottage cheese (8.4% fat). The candidate method was compared to the ISO 11290–1/Amd.1 reference method. Sixteen participants from 15 laboratories throughout the European Union participated. Three levels of contamination were evaluated: a non-inoculated control level (0 CFU/test portion), a low contamination level (∼2 CFU/test portion), and a high contamination level (∼10 CFU/test portion). Data from that study were analyzed according to the probability of detection (POD) statistical model. Results The dLPODC values with 95% confidence intervals between the candidate and reference method results were −0.02 (−0.07, 0.03), −0.08 (−0.31, 0.16), and 0.00 (−0.03, 0.03) for the non-inoculated, low, and high contamination levels, respectively. Conclusions The dLPODC results demonstrate no difference in performance between the candidate method and reference method for the matrix evaluated. Highlights Data from a singular collaborative study was used to achieve adoption as an AOAC First Action Official Method for the detection of Listeria species in a variety of foods and select environmental surfaces.

Objective To describe the clinical presentation, treatment, and clinical outcome of horses with ocular disease and evidence of systemic or ocular Lyme disease. Animals studied Five horses met the inclusion criteria of ocular disease with evidence of B burgdorferi present in ocular or CNS tissues. Procedure The goal of this study was to describe the clinical presentation and progression of ocular disease when associated with ocular or CNS B burgdorferi infection in horses. A retrospective review of medical records was performed on horses admitted for ocular disease with evidence of B burgdorferi infection between 1998 and 2015. The diagnosis of B burgdorferi-associated uveitis was based on histopathologic lesions of lymphohistiocytic and suppurative uveitis/endophthalmitis and intralesional argyrophilic spirochetes in either ocular or CNS tissue consistent with Borrelia. Leptospiral uveitis was ruled out by PCR. Results All five horses in the current study had intraocular inflammation at the time of presentation. Medical management with anti-inflammatories was successful in controlling uveitis in the two horses in which treatment of uveitis was attempted. Systemic treatment with oral tetracyclines was unsuccessful in a single case in which treatment of Borrelia was attempted. Four horses were euthanized due to progression of neurologic disease. The surviving horse had an enucleation performed and did not show systemic signs. Conclusions Infection with Borrelia burgdorferi should be considered in endemic areas as a differential for horses with ocular disease, in particular, uveitis. The prognosis for uveitis and neurologic disease associated with Lyme disease was poor in the current study.

Mechanisms driving tumor progression from less aggressive subtypes to more aggressive states represent key targets for therapy. We identified a subset of luminal A primary breast tumors that give rise to HER2-enriched (HER2E) subtype metastases, but remain clinically HER2 negative (cHER2(–)). By testing the unique genetic and transcriptomic features of these cases, we developed the hypothesis that FGFR4 likely participates in this subtype switching. To evaluate this, we developed 2 FGFR4 genomic signatures using a patient-derived xenograft (PDX) model treated with an FGFR4 inhibitor, which inhibited PDX growth in vivo. Bulk tumor gene expression analysis and single-cell RNA sequencing demonstrated that the inhibition of FGFR4 signaling caused molecular switching. In the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) breast cancer cohort, FGFR4-induced and FGFR4-repressed signatures each predicted overall survival. Additionally, the FGFR4-induced signature was an independent prognostic factor beyond subtype and stage. Supervised analysis of 77 primary tumors with paired metastases revealed that the FGFR4-induced signature was significantly higher in luminal/ER(+) tumor metastases compared with their primaries. Finally, multivariate analysis demonstrated that the FGFR4-induced signature also predicted site-specific metastasis for lung, liver, and brain, but not for bone or lymph nodes. These data identify a link between FGFR4-regulated genes and metastasis, suggesting treatment options for FGFR4-positive patients, whose high expression is not caused by mutation or amplification.

Identifying modifiers of dosage-sensitive genes involved in neurodegenerative disorders is imperative to discover novel genetic risk factors and potential therapeutic entry points. In this study, we focus on Ataxin-1 (ATXN1), a dosage-sensitive gene involved in the neurodegenerative disease spinocerebellar ataxia type 1 (SCA1). While the precise maintenance of ATXN1 levels is essential to prevent disease, the mechanisms that regulate ATXN1 expression remain largely unknown. We demonstrate that ATXN1’s unusually long 5′ untranslated region (5′ UTR) negatively regulates its expression via posttranscriptional mechanisms. Based on recent reports that microRNAs (miRNAs) can interact with both 3′ and 5′ UTRs to regulate their target genes, we identify miR760 as a negative regulator that binds to a conserved site in ATXN1’s 5′ UTR to induce RNA degradation and translational inhibition. We found that delivery of Adeno-associated virus (AAV)-expressing miR760 in the cerebellum reduces ATXN1 levels in vivo and mitigates motor coordination deficits in a mouse model of SCA1. These findings provide new insights into the regulation of ATXN1 levels, present additional evidence for miRNA-mediated gene regulation via 5′ UTR binding, and raise the possibility that noncoding mutations in the ATXN1 locus may act as risk factors for yet to be discovered progressive ataxias.

The timing of reproduction is often governed by environmental variables, such as temperature or rainfall. Understanding how environmental variables affect mating dynamics is necessary to predict how systems and populations may adapt to changing environmental conditions and is crucial for management of threatened species. The American horseshoe crab (Limulus polyphemus) ranges from the Yucatan to Maine in distinct populations that differ in their timing of reproduction; while most populations have only one breeding period during the spring, some southern populations have two breeding periods. Here we discuss seasonal patterns of reproduction in a Florida Gulf coast population where horseshoe crabs have two periods of breeding: one in the spring and another in the fall. We used environmental measurements, spawning surveys, mark-recapture, and measurements of adult traits and spawning behavior to compare reproductive parameters between the two spawning seasons over three years. We then evaluated whether environmental conditions affect fall and spring horseshoe crab nesting patterns similarly and whether fall and spring horseshoe crabs should be considered two separate populations. We found significant differences in environmental conditions across seasons and in a wide variety of horseshoe crab traits and nesting parameters. Furthermore, environmental conditions affected nesting behaviors of fall and spring horseshoe crabs differently. However, some individuals spawn during both seasons, suggesting that trait differences may be attributable to environmental effects during development or seasonal plasticity, rather than genetic differences, although further study is necessary. Finally, our results suggest that management practices should be tailored to each population, because environmental conditions may have different effects even on genetically similar groups.