The Drosophila spectraplakin Short stop regulates focal adhesion dynamics by cross-linking microtubules and actin.

The spectraplakin family of proteins includes ACF7/MACF1 and BPAG1/dystonin in mammals, VAB-10 in Caenorhabditis elegans, Magellan in zebrafish, and Short stop (Shot), the sole Drosophila member. Spectraplakins are giant cytoskeletal proteins that cross-link actin, microtubules, and intermediate filaments, coordinating the activity of the entire cytoskeleton. We examined the role of Shot during cell migration using two systems: the in vitro migration of Drosophila tissue culture cells and in vivo through border cell migration. RNA interference (RNAi) depletion of Shot increases the rate of random cell migration in Drosophila tissue culture cells as well as the rate of wound closure during scratch-wound assays. This increase in cell migration prompted us to analyze focal adhesion dynamics. We found that the rates of focal adhesion assembly and disassembly were faster in Shot-depleted cells, leading to faster adhesion turnover that could underlie the increased migration speeds. This regulation of focal adhesion dynamics may be dependent on Shot being in an open confirmation. Using Drosophila border cells as an in vivo model for cell migration, we found that RNAi depletion led to precocious border cell migration. Collectively, these results suggest that spectraplakins not only function to cross-link the cytoskeleton but may regulate cell-matrix adhesion.

S1 guidelines "lumbar puncture and cerebrospinal fluid analysis" (abridged and translated version).

INTRODUCTION: Cerebrospinal fluid (CSF) analysis is important for detecting inflammation of the nervous system and the meninges, bleeding in the area of the subarachnoid space that may not be visualized by imaging, and the spread of malignant diseases to the CSF space. In the diagnosis and differential diagnosis of neurodegenerative diseases, the importance of CSF analysis is increasing. Measuring the opening pressure of CSF in idiopathic intracranial hypertension and at spinal tap in normal pressure hydrocephalus constitute diagnostic examination procedures with therapeutic benefits.Recommendations (most important 3-5 recommendations on a glimpse): The indications and contraindications must be checked before lumbar puncture (LP) is performed, and sampling CSF requires the consent of the patient.Puncture with an atraumatic needle is associated with a lower incidence of postpuncture discomfort. The frequency of postpuncture syndrome correlates inversely with age and body mass index, and it is more common in women and patients with a history of headache. The sharp needle is preferably used in older or obese patients, also in punctures expected to be difficult.In order to avoid repeating LP, a sufficient quantity of CSF (at least 10 ml) should be collected. The CSF sample and the serum sample taken at the same time should be sent to a specialized laboratory immediately so that the emergency and basic CSF analysis program can be carried out within 2 h.The indication for LP in anticoagulant therapy should always be decided on an individual basis. The risk of interrupting anticoagulant therapy must be weighed against the increased bleeding risk of LP with anticoagulant therapy.As a quality assurance measure in CSF analysis, it is recommended that all cytological, clinical-chemical, and microbiological findings are combined in an integrated summary report and evaluated by an expert in CSF analysis. CONCLUSIONS: In view of the importance and developments in CSF analysis, the S1 guideline "Lumbar puncture and cerebrospinal fluid analysis" was recently prepared by the German Society for CSF analysis and clinical neurochemistry (DGLN) and published in German in accordance with the guidelines of the AWMF (https://www.awmf.org). /uploads/tx_szleitlinien/030-141l_S1_Lumbalpunktion_und_Liquordiagnostik_2019-08.pdf). The present article is an abridged translation of the above cited guideline. The guideline has been jointly edited by the DGLN and DGN.

Effect of a post-hatch lipopolysaccharide challenge in Turkey poults and ducklings after a primary embryonic heat stress.

The effect of embryonic thermal manipulation on the post-hatch immune response to a lipopolysaccharide (LPS) challenge was studied in Pekin ducklings and turkey poults. Commercial duck and turkey eggs were distributed among four treatments: SS-Control (37.5 °C from embryonic day [ED] 1 to 25); SS-LPS (37.5 °C from ED1 to 25 + LPS at D0 [hatch]); HH-LPS (38 °C from ED1 to 25 + LPS at D0; SH-LPS (37.5 °C from ED1 to 10 and 38 °C from ED 11 to 25 + LPS at D0). At ED16 and ED24, the egg shell temperature of the duck and turkey eggs in the HH and SH treatments were higher (P ≤ 0.01) than the SS treatment. Ducklings and poults in the HH treatment had the lowest yolk free body weight at hatch (P ≤ 0.05). At 24, 48, and 72 h post-LPS injection, ducklings and poults in the HH-LPS treatment had significantly reduced BW compared with the SS-Con treatments (P ≤ 0.05). Ducklings and poults in the SH-LPS and HH-LPS treatments had increased plasma heat shock protein 70 (HSP70) and lower splenic HSP70 mRNA amounts than the SS-LPS treatments at 24, and 48 h post-challenge (P ≤ 0.05). At 48 and 72 h, macrophage nitric oxide (NO) production in ducklings and poults in the SH-LPS and HH-LPS treatments was lower than in the SS-LPS treatments (P ≤ 0.05). Ducklings and poults in the SH-LPS treatment had increased thymocyte proliferation compared to the SS-LPS treatment at 24, 48 and 72 h (P ≤ 0.05). At 24 h, ducklings in the SH-LPS treatment had increased splenic IL-10 and reduced IFNγ and IL-6 mRNA abundance. However, both ducklings and poults in the HH-LPS treatment had increased IFNγ, and IL-10 mRNA abundance compared to the SS-LPS treatment (P ≤ 0.05). At 48 h, SH-LPS ducklings and poults had lower splenic IL-10 mRNA abundance (P ≤ 0.05) while the HH-LPS treatment resulted in comparable splenic IL-10 mRNA compared to the SS-LPS treatment (P ≥ 0.05). Ducklings and poults in the SH-LPS treatment had increased thymic and splenic CD8+/CD4+ ratios at 24 h versus the SS-LPS treatment (P ≤ 0.05). In conclusion, embryonic thermal manipulation from ED11-25 increased extracellular HSP70 release, thymocyte proliferation and IL-10 but decreased splenic HSP70 and IFNγ mRNA amounts at 24 h post-LPS injection. This suggests that mild heat stress during the later stages of incubation could potentially prime the embryonic immune system thereby enhances the immune response as earlier than 24 h to eliminate the inflammatory response without affecting the growth performance by increase the extracellular release of HSP70 in both ducklings and poults. Continuous exposure to the small increase in temperature from ED 1-25 (HH) caused an imbalance between pro (IFNγ)- and anti-inflammatory cytokines(IL-10) which affects hatchling responses to an inflammatory challenge and increased mortality. The amount of extracellular HSP70 could potentially play an important role in modulating the immune response against inflammatory challenges.

Effect of embryonic thermal manipulation on heat shock protein 70 (HSP70) expression and subsequent immune response to post-hatch lipopolysaccharide challenge in Pekin ducklings.

During the course of multi-stage incubation, small locational differences in incubation temperature within a machine are not uncommon and so the goal of this study was to study the immune response of ducklings exposed to thermal manipulation during incubation. Commercial Pekin duck eggs (n = 200) were distributed among four treatment: SS-Control (37.5°C from embryonic day [ED] 1 to 25); SS-LPS (37.5°C from ED1 to 25 + LPS at D0 [hatch]); HH-LPS (38°C from ED1 to 25+ LPS at D0); SH-LPS (37.5°C from ED1 to 10 and 38°C from ED 11 to 25 + LPS at D0). At D0, ducklings received a lipopolysaccharide (LPS) injection. At D1 and D5, the HH-LPS treatment significantly reduced body weight (P ≤ 0.05). At D1 and D3 post-LPS injection, the SH-LPS and HH-LPS treatments significantly reduced splenic and bursal heat shock protein 70 (HSP70), mRNA abundance, and macrophage nitric oxide production compared with the SS-LPS treatment (P ≤ 0.05). At D1, the HH-LPS and SH-LPS treatments had increased splenic IL-10 mRNA and lower MHC I mRNA compared with the SS-LPS treatment (P ≤ 0.05). At D1, the HH-LPS treatment increased splenic IL-6 mRNA and bursal IFNγ mRNA transcription while the SH-LPS treatment reduced splenic IL-6 mRNA compared with the SS-LPS treatment (P ≤ 0.05). The HH-LPS treatment reduced thymocyte proliferation efficiency, while at D1, D3, and D5, the SH-LPS treatment increased thymocyte proliferation efficiency compared with the SS-LPS treatment (P ≤ 0.05). Ducklings in the HH-LPS treatment had a higher splenic CD8+/CD4+ ratio compared to the SS-LPS treatment at D3 post-LPS injection (P ≤ 0.05). In summary, the HH-LPS treatment compromised immunocompetence via decreased NO production and thymocyte proliferation efficiency, while the SH-LPS treatment increased body weight and thymocyte proliferation and reduced IL-6 mRNA abundance. This suggests that an embryonic temperature stress during the latter half of incubation may prime the immune system which may be beneficial during secondary post-hatch inflammatory challenges.

From muscle to food: oxidative challenges and developmental anomalies in poultry breast muscle.

The glycolytic capacity in the "white muscle fibers" found predominantly in broiler and turkey breast muscle have long been recognized as being key components contributing to optimal pH changes post-harvest and the quality of breast muscle products. The pectoralis major (PMAJ) is the predominant breast muscle and is almost exclusively comprised of fast-twitch glycolytic (anaerobic) type IIb muscle fibers (white). In nature, the pectoralis muscles are designed to meet the intense, periodic energy demands of flight in contrast to the continual, oxidative metabolism (aerobic) inherent to the type I slow twitch fibers (red) in the thigh and drum. Within the past decade, it has been recognized that increased breast muscle yield in modern broiler strains may have contributed to muscle myopathies characterized by visible white striations (white striping; WS) often accompanied by surface hemorrhages and areas of extreme firmness (woody breast; WB). The research to date has primarily addressed differences in muscle physiology and processing characteristics in "normal" and "abnormal" muscle samples from older broilers. One hypothesis is that the extensive accretion of protein in a poorly vascularized breast muscle overwhelms the capacity for coping with excessive production of reactive oxygen species and the term "oxidative stress" has been used to describe this condition. There are few recent reports in poultry, however, which have addressed the potential negative physiological effects imposed by multiple layers of fascia surrounding individual muscle fibers, fiber bundles, and intact muscles on circulation and oxidative stability. The objective of this review is to support our hypothesis that the extensive accretion of breast muscle in a poorly vascularized and tightly bound PMAJ could be creating a "compartment syndrome" like condition leading to the development of the WS and WB anomalies in heavy, high yielding birds. Recent gene expression data support this hypothesis.

Transcriptional comparison of myogenesis in leghorn and low score normal embryos.

In a previous study, we reported on a quantitative transcriptomic method which confirmed the temporal transcription of developmental fast skeletal muscle myosin heavy chain (fsMyHC) embryonic isoforms in the embryonic Pectoralis major (PM) of the Single Comb White Leghorn (SCWL). The objective of the current study was to further investigate the transcriptional events underlying embryonic PM growth in the SCWL and a genotype exhibiting partial muscular dystrophy, the Low Score Normal (LSN). We hypothesized that within the SCWL and LSN embryos, there would be differences in the temporal transcription of the fsMyHC isoforms and other myogenic regulatory genes. Samples of PM tissues were collected daily from embryonic day (ED) 6 through ED19. Total RNA was isolated from each PM tissue sample and mRNA transcripts from 28 target genes were simultaneously quantified using a probe hybridization method. Raw data counts were normalized against the geometric mean of 5 housekeeping genes and analyzed using Local Regression (LOESS) smoothing methods. Predicted estimates based on LOESS smoothing were plotted with 95% upper and lower confidence intervals, allowing for line comparisons between the SCWL and LSN. Differences (P < 0.05) were determined by non-overlapping confidence intervals between the SCWL and LSN. Among genes exhibiting line differences (P < 0.05), were the developmental fsMyHC isoforms, transcription factors, growth factors, and proteoglycans. The current study is the first to report transcription of the chicken fast skeletal adult myosin isoform (Cad) during PM myogenesis. Samples were submitted for capillary-liquid chromatography-tandem mass spectrometry analysis which confirmed the translation of all the developmental fsMyHC isoforms including Cad in both lines. The LSN exhibited altered transcription patterns of early myogenic markers (MYOD, MRF4, Cemb1, six4, and pax3) during late embryogenesis, continued transcription of Cvent, and delayed transcription of Cneo.

Motor Neurons Tune Premotor Activity in a Vertebrate Central Pattern Generator.

Central patterns generators (CPGs) are neural circuits that drive rhythmic motor output without sensory feedback. Vertebrate CPGs are generally believed to operate in a top-down manner in which premotor interneurons activate motor neurons that in turn drive muscles. In contrast, the frog (Xenopus laevis) vocal CPG contains a functionally unexplored neuronal projection from the motor nucleus to the premotor nucleus, indicating a recurrent pathway that may contribute to rhythm generation. In this study, we characterized the function of this bottom-up connection. The X. laevis vocal CPG produces a 50-60 Hz "fast trill" song used by males during courtship. We recorded "fictive vocalizations" in the in vitro CPG from the laryngeal nerve while simultaneously recording premotor activity at the population and single-cell level. We show that transecting the motor-to-premotor projection eliminated the characteristic firing rate of premotor neurons. Silencing motor neurons with the intracellular sodium channel blocker QX-314 also disrupted premotor rhythms, as did blockade of nicotinic synapses in the motor nucleus (the putative location of motor neuron-to-interneuron connections). Electrically stimulating the laryngeal nerve elicited primarily IPSPs in premotor neurons that could be blocked by a nicotinic receptor antagonist. Our results indicate that an inhibitory signal, activated by motor neurons, is required for proper CPG function. To our knowledge, these findings represent the first example of a CPG in which precise premotor rhythms are tuned by motor neuron activity.SIGNIFICANCE STATEMENT Central pattern generators (CPGs) are neural circuits that produce rhythmic behaviors. In vertebrates, motor neurons are not commonly known to contribute to CPG function, with the exception of a few spinal circuits where the functional significance of motor neuron feedback is still poorly understood. The frog hindbrain vocal circuit contains a previously unexplored connection from the motor to premotor region. Our results indicate that motor neurons activate this bottom-up connection, and blocking this signal eliminates normal premotor activity. These findings may promote increased awareness of potential involvement of motor neurons in a wider range of CPGs, perhaps clarifying our understanding of network principles underlying motor behaviors in numerous organisms, including humans.

The MRZ reaction as a highly specific marker of multiple sclerosis: re-evaluation and structured review of the literature.

BACKGROUND: It has long been known that the majority of patients with multiple sclerosis (MS) display an intrathecal, polyspecific humoral immune response to a broad panel of neurotropic viruses. This response has measles virus, rubella virus and varicella zoster virus as its most frequent constituents and is thus referred to as the MRZ reaction (MRZR). OBJECTIVE: Re-evaluation of the specificity of MRZR as a marker of MS. METHODS: Structured review of the existing English-, German- and Spanish-language literature on MRZR testing, with evaluation of MRZR in a cohort of 43 unselected patients with MS and other neurological diseases as a proof of principle. RESULTS: A positive MRZ reaction, defined as a positive intrathecal response to at least two of the three viral agents, was found in 78% of MS patients but only in 3% of the controls (p < 0.00001), corresponding to specificity of 97%. Median antibody index values were significantly lower in non-MS patients (measles, p < 0.0001; rubella, p < 0.006; varicella zoster, p < 0.02). The 30 identified original studies on MRZR reported results from 1478 individual MRZR tests. A positive MRZR was reported for 458/724 (63.3%) tests in patients with MS but only for 19/754 (2.5%) tests in control patients (p < 0.000001), corresponding to cumulative specificity of 97.5% (CI 95% 96-98.4), cumulative sensitivity of 63.3% (CI 95% 59.6-66.8) (or 67.4% [CI 95% 63.5-71.1] in the adult MS subgroup), a positive likelihood ratio of 25.1 (CI 95% 16-39.3) and a negative likelihood ratio of 0.38 (CI 95% 0.34-0.41). Of particular note, MRZR was absent in 52/53 (98.1%) patients with neuromyelitis optica or MOG-IgG-positive encephalomyelitis, two important differential diagnoses of MS. CONCLUSION: MRZR is the most specific laboratory marker of MS reported to date. If present, MRZR substantially increases the likelihood of the diagnosis of MS. Prospective and systematic studies on the diagnostic and prognostic impact of MRZR testing are highly warranted.

[Cytology of cerebrospinal fluid : Standards, importance and modern methods]. / Liquorzytologie : Standards, Stellenwert und moderne Methoden.

Cytology is an integral part of cerebrospinal fluid (CSF) analysis. It is relevant for the diagnostics and differential diagnosis of inflammatory, hemorrhagic and neoplastic central nervous system (CNS) processes. This article summarizes the recommended procedures and typical clinical patterns. In addition, modern immunocytochemical and flow cytometry methods for CSF cytology are presented. In particular, the diagnostic contribution and clinical relevance in several CNS conditions are discussed.

Early intestinal development and mucin transcription in the young poult with probiotic and mannan oligosaccharide prebiotic supplementation.

Alternative and adjunctive approaches to decreasing the use of dietary antibiotics are becoming popular areas of study. Supplemental probiotics (commensal microbes) and prebiotics (indigestible complex carbohydrates) are 2 dietary approaches to facilitating the intestinal colonization of beneficial bacteria to compete with potential pathogens, thus creating a healthy mucosal environment. The intestinal mucosa is composed of mucin glycoproteins, which play a key role in preventing the attachment of pathogenic bacteria. At hatch, the neonatal turkey intestine is relatively aseptic and vulnderable to bacterial colonization by both commensal and pathogenic microbes. In the current study, we determined the transcription of MUC2, the primary mucin protein produced by goblet cells within the small intestine, and we also measured intestinal morphology immediately post-hatch through d 11. Poults were fed a conventional starter diet, the starter diet supplemented with one of 2 commercial probiotics (A, B), or a commercial mannan oligosaccharide. MUC2 transcription increased from d zero to d 4 post-hatch (P< 0.05), but there was no effect of probiotic or prebiotic supplementation. Villus height and villus area both increased with Probiotic B and mannan oligosaccharide supplementation (P<0.05) and there was a significant d X treatment interaction effect for crypt depth (P=0.007). These results suggest that probiotic and prebiotic supplementation can positively alter the intestinal microenvironment.

Temporal embryonic transcription of chicken fast skeletal myosin heavy chain isoforms in the single comb white leghorn.

There are numerous factors that can significantly influence embryonic development in poultry and thus make simple days of incubation (chronological age) a less than perfect metric for studying embryonic physiology. The developmental fast skeletal muscle myosin (MyHC), the predominant protein in the Pectoralis major (PM), is temporally expressed as a cadre of highly specific developmental isoforms. In the study described herein, a novel molecular technology (NanoString) was used to characterize the myosin isoform transcriptional patterns in the PM of Single Comb White Leghorn (SCWL) embryos. NanoString technology is based on quantitative analysis of the transcriptome through digital detection and quantification of target mRNA transcripts. Total RNA was isolated and gene transcription quantified using NanoString in embryonic muscle samples collected daily from 6 through 19 days of incubation. Data were analyzed using the LOESS smoothing function at a 95% confidence level. The temporal transcription of MyHC isoforms obtained in this study was consistent with the literature at higher specificity and resolution, thus validating NanoString for use in gene transcription analyses. The results support a hypothesis that the transcription patterns of the embryonic MyHC isoforms may be used as molecular clocks to further investigate the developmental relationships underlying embryonic fast skeletal muscle growth and development.

The Gas2 family protein Pigs is a microtubule +TIP that affects cytoskeleton organisation.

Coordination between different cytoskeletal systems is crucial for many cell biological functions, including cell migration and mitosis, and also plays an important role during tissue morphogenesis. Proteins of the class of cytoskeletal crosslinkers, or cytolinkers, have the ability to interact with more than one cytoskeletal system at a time and are prime candidates to mediate any coordination. One such class comprises the Gas2-like proteins, combining a conserved calponin-homology-type actin-binding domain and a Gas2 domain predicted to bind microtubules (MTs). This domain combination is also found in spectraplakins, huge cytolinkers that play important roles in many tissues in both invertebrates and vertebrates. Here, we dissect the ability of the single Drosophila Gas2-like protein Pigs to interact with both actin and MT cytoskeletons, both in vitro and in vivo, and illustrate complex regulatory interactions that determine the localisation of Pigs to and its effects on the cytoskeleton.

Does MIPO of fractures of the distal femur result in more rotational malalignment than ORIF? A retrospective study.

PURPOSE: Intraoperative control of rotational malalignment poses a big challenge for surgeons when using modern MIPO (minimally invasive plate osteosynthesis) techniques. We hypothesized that distal femoral fractures treated with MIPO technique are more often fixed in malrotation than those treated with open reduction internal fixation (ORIF). METHODS: In this retrospective study, we identified 20 patients who met the inclusion criteria and agreed to take part in the study. In ten patients MIPO was applied, in the other ten ORIF was used. Mean age was 44.8 (19-71 years). Functional status was assessed using clinical scores (Harris Hip Score, WOMAC Hip, KS Score, WOMAC Knee, Kujala Score). Rotational alignment was assessed with magnetic resonance imaging and compared to the opposite leg. RESULTS: We discovered a significant difference in the mean rotational difference between the MIPO group (14.3°) and the ORIF group (5.2°). Functionally, patients in the ORIF group outperformed patients in the MIPO group in all clinical scoring systems although no one proved to be statistically significant. MIPO technique was associated with significantly more rotational malalignment compared to ORIF in distal femur fracture fixation. However, implant failure and nonunion was more common in the ORIF group, with a revision rate of 3 versus 1 in the ORIF group. Clinical scoring did not significantly different between both groups. CONCLUSION: Taking into account the undisputable advantages of minimally invasive surgery, improved teaching of methods to avoid malrotation as well as regular postoperative investigations to detect any malrotation should be advocated.

Macrophage and dendritic cell subsets in IBD: ALDH+ cells are reduced in colon tissue of patients with ulcerative colitis regardless of inflammation.

Disruption of the homeostatic balance of intestinal dendritic cells (DCs) and macrophages (MQs) may contribute to inflammatory bowel disease. We characterized DC and MQ populations, including their ability to produce retinoic acid, in clinical material encompassing Crohn's ileitis, Crohn's colitis and ulcerative colitis (UC) as well as mesenteric lymph nodes (MLNs) draining these sites. Increased CD14(+)DR(int) MQs characterized inflamed intestinal mucosa while total CD141(+) or CD1c(+) DCs numbers were unchanged. However, CD103(+) DCs, including CD141(+)CD103(+) and CD1c(+)CD103(+) DCs, were reduced in inflamed intestine. In MLNs, two CD14(-) DC populations were identified: CD11c(int)HLADR(hi) and CD11c(hi)HLADR(int) cells. A marked increase of CD11c(hi)HLADR(int) DC, particularly DR(int)CD1c(+) DCs, characterized MLNs draining inflamed intestine. The fraction of DC and MQ populations expressing aldehyde dehydrogenase (ALDH) activity, reflecting retinoic acid synthesis, in UC colon, both in active disease and remission, were reduced compared to controls and inflamed Crohn's colon. In contrast, no difference in the frequency of ALDH(+) cells among blood precursors was detected between UC patients and non-inflamed controls. This suggests that ALDH activity in myeloid cells in the colon of UC patients, regardless of whether the disease is active or in remission, is influenced by the intestinal environment.

EULAR recommendations for the use of imaging in the diagnosis and management of spondyloarthritis in clinical practice.

A taskforce comprised of an expert group of 21 rheumatologists, radiologists and methodologists from 11 countries developed evidence-based recommendations on the use of imaging in the clinical management of both axial and peripheral spondyloarthritis (SpA). Twelve key questions on the role of imaging in SpA were generated using a process of discussion and consensus. Imaging modalities included conventional radiography, ultrasound, magnetic resonance imaging, computed tomography (CT), positron emission tomography, single photon emission CT, dual-emission x-ray absorptiometry and scintigraphy. Experts applied research evidence obtained from systematic literature reviews using MEDLINE and EMBASE to develop a set of 10 recommendations. The strength of recommendations (SOR) was assessed by taskforce members using a visual analogue scale. A total of 7550 references were identified in the search process, from which 158 studies were included in the systematic review. Ten recommendations were produced using research-based evidence and expert opinion encompassing the role of imaging in making a diagnosis of axial SpA or peripheral SpA, monitoring inflammation and damage, predicting outcome, response to treatment, and detecting spinal fractures and osteoporosis. The SOR for each recommendation was generally very high (range 8.9-9.5). These are the first recommendations which encompass the entire spectrum of SpA and evaluate the full role of all commonly used imaging modalities. We aimed to produce recommendations that are practical and valuable in daily practice for rheumatologists, radiologists and general practitioners.

Eukaryotic inhibitors or activators elicit responses to chemosensory compounds by ruminal isotrichid and entodiniomorphid protozoa.

Our objectives were to evaluate potential signaling pathways regulating rumen protozoal chemotaxis using eukaryotic inhibitors potentially coordinated with phagocytosis as assessed by fluorescent bead uptake kinetics. Wortmannin (inhibitor of phosphoinositide 3-kinase), insulin, genistein (purported inhibitor of a receptor tyrosine kinase), U73122 (inhibitor of phospholipase C), and sodium nitroprusside (Snp, nitric oxide generator, activating protein kinase G) were preincubated with mixed ruminal protozoa for 3h before assessing uptake of fluorescent beads and chemosensory behavior to glucose, peptides, and their combination; peptides were also combined with guanosine triphosphate (GTP; a chemorepellent). Entodiniomorphids were chemoattracted to both glucose and peptides, but chemoattraction to glucose was increased by Snp and wortmannin without effect on chemoattraction to peptides. Rate of fluorescent bead uptake by an Entodinium caudatum culture decreased when beads were added simultaneously with feeding and incubated with wortmannin (statistical interaction). Wortmannin also decreased the proportion of mixed entodiniomorphids consuming beads. Isotrichid protozoa exhibited greater chemotaxis to glucose but, compared with entodiniomorphids, were chemorepelled to peptides. Wortmannin increased chemotaxis by entodiniomorphids but decreased chemotaxis to glucose by isotrichids. Motility assays documented that Snp and wortmannin decreased net swimming speed (distance among 2 points per second) but not total swimming speed (including turns) by entodiniomorphids. Wortmannin decreased both net and total swimming behavior in isotrichids. Results mechanistically explain the isotrichid migratory ecology to rapidly take up newly ingested sugars and subsequent sedimentation back to the ventral reticulorumen. In contrast, entodiniomorphids apparently integrate cellular motility with feeding behavior to consume small particulates and thereby stay associated and pass with the degradable fraction of rumen particulates. These results extend findings from aerobic ciliate models to explain how rumen protozoa have adapted physiology for their specific ecological niches.

Clinical characteristics of ovarian cancer classified by BRCA1, BRCA2, and RAD51C status.

We evaluated homologous recombination deficient (HRD) phenotypes in epithelial ovarian cancer (EOC) considering BRCA1, BRCA2, and RAD51C in a large well-annotated patient set. We evaluated EOC patients for germline deleterious mutations (n = 899), somatic mutations (n = 279) and epigenetic alterations (n = 482) in these genes using NGS and genome-wide methylation arrays. Deleterious germline mutations were identified in 32 (3.6%) patients for BRCA1, in 28 (3.1%) for BRCA2 and in 26 (2.9%) for RAD51C. Ten somatically sequenced patients had deleterious alterations, six (2.1%) in BRCA1 and four (1.4%) in BRCA2. Fifty two patients (10.8%) had methylated BRCA1 or RAD51C. HRD patients with germline or somatic alterations in any gene were more likely to be high grade serous, have an earlier diagnosis age and have ovarian and/or breast cancer family history. The HRD phenotype was most common in high grade serous EOC. Identification of EOC patients with an HRD phenotype may help tailor specific therapies.

Second-trimester diagnosis of triploidy: a series of four cases.

Triploidy occurs in 2 to 3% of conceptuses and accounts for approximately 20% of chromosomally abnormal first-trimester miscarriages. As such, triploidy is estimated to occur in 1 of 3,500 pregnancies at 12 weeks', 1 in 30,000 at 16 weeks', and 1 in 250,000 at 20 weeks' gestation. We present a series of four cases of second-trimester triploidy diagnosed at our center within a 1-year timeframe. This is remarkable, as the delivery volume at our institution is roughly 2,500/y. All patients were at least 19 weeks' gestation, with multiple abnormalities identified on prenatal ultrasound at 18 to 20 weeks' gestation; all fetuses had lethal anomalies, but anomalies were not consistent between cases. All patients elected for induction of labor before 24 weeks' gestational age. Two of the four cases had amniocentesis and chromosome analysis prior to delivery, and two cases had chromosome analysis performed on fetal tissue after delivery. All fetuses were examined following delivery. This case series demonstrates that the diagnosis of triploidy may not be obvious based on ultrasound and physical examination findings and highlights the importance of routine chromosome analysis on all prenatal diagnoses of multiple congenital anomalies prior to consideration of more complex genetic testing.