Modes of action of the archaeal Mre11/Rad50 DNA-repair complex revealed by fast-scan atomic force microscopy.

Mre11 and Rad50 (M/R) proteins are part of an evolutionarily conserved macromolecular apparatus that maintains genomic integrity through repair pathways. Prior structural studies have revealed that this apparatus is extremely dynamic, displaying flexibility in the long coiled-coil regions of Rad50, a member of the structural maintenance of chromosome (SMC) superfamily of ATPases. However, many details of the mechanics of M/R chromosomal manipulation during DNA-repair events remain unclear. Here, we investigate the properties of the thermostable M/R complex from the archaeon Sulfolobus acidocaldarius using atomic force microscopy (AFM) to understand how this macromolecular machinery orchestrates DNA repair. While previous studies have observed canonical interactions between the globular domains of M/R and DNA, we observe transient interactions between DNA substrates and the Rad50 coiled coils. Fast-scan AFM videos (at 1-2 frames per second) of M/R complexes reveal that these interactions result in manipulation and translocation of the DNA substrates. Our study also shows dramatic and unprecedented ATP-dependent DNA unwinding events by the M/R complex, which extend hundreds of base pairs in length. Supported by molecular dynamic simulations, we propose a model for M/R recognition at DNA breaks in which the Rad50 coiled coils aid movement along DNA substrates until a DNA end is encountered, after which the DNA unwinding activity potentiates the downstream homologous recombination (HR)-mediated DNA repair.

Retrospective evaluation of canine primary, multicentric, and metastatic intraocular neoplasia.

OBJECTIVE: To provide an updated characterization of the prevalence of primary, multicentric, and metastatic intraocular tumors in the canine patient. PROCEDURES: Medical records databases from 4 veterinary referral hospitals were reviewed from 1999 to present to identify dogs with a diagnosis of intraocular neoplasia histopathologically confirmed following enucleation or necropsy. RESULTS: One hundred seventy-two dogs with 173 intraocular neoplasms met the inclusion criteria. Primary intraocular neoplasms were the most common tumors in the study (128); the two most common types were melanocytic neoplasia (90), followed by iridociliary neoplasia (33). There were 28 cases of intraocular involvement secondary to round cell neoplasia, with 18 cases of lymphoma, seven histiocytic sarcomas, and three undifferentiated round cell neoplasms. There were 17 cases of metastatic intraocular neoplasia, with hemangiosarcoma being the most common (9). CONCLUSIONS: The majority of intraocular tumors in dogs arise from the ocular tissues. However, the eye may also be involved in patients with multicentric neoplasia, and, less commonly, as a site for metastatic disease. Ocular screening for patients with multicentric neoplasia should be considered during staging, and ocular signs should be viewed with suspicion in dogs with neoplasia in other sites.

Effects of stent postdilatation during primary PCI for STEMI: Insights from coronary physiology and optical coherence tomography.

OBJECTIVES: This study aimed to assess the impact of stent optimization by NC-balloon postdilatation (PD) during primary-PCI for STEMI with the use of coronary physiology and intracoronary imaging. METHODS: This was a prospective observational study (ClinicalTrials.gov:NCT02788396). Optical coherence tomography (OCT) and physiological measurements were performed immediately before and after PD with the operators blinded to all measurements. The index of microcirculatory resistance (IMR), coronary flow reserve (CFR) and fractional flow reserve (FFR) were measured. OCT analysis was performed for assessment of stent expansion, malapposition, in-stent plaque-thrombus prolapse (PTP) and stent-edge dissections (SED). The change in IMR before and after PD as a measure of microvascular injury was the primary objective of the study. RESULTS: Thirty-two STEMI patients undergoing primary-PCI had physiological measurements before and after PD. All patients received second-generation DES (diameter 3.1 ± 0.5 mm, length 29.9 ± 10.7 mm) and postdilatation with NC-balloons (diameter 3.6 ± 0.6 mm, inflation pressure 19.3 ± 2.0 atm). IMR (44.9 ± 25.6 vs. 48.8 ± 34.2, p = 0.26) and CFR (1.60 ± 0.89 vs. 1.58 ± 0.71, p = 0.87) did not change, while FFR increased after PD (0.91 ± 0.08 vs. 0.93 ± 0.06, p = 0.037). At an individual patient level, IMR increased in half of the cases. PD improved significantly absolute and relative stent expansion, reduced malapposition, and increased PTP. There was no difference in clinically relevant SED. CONCLUSION: In this exploratory, hypothesis-generating study, postdilatation during primary-PCI for STEMI improved stent expansion, apposition and post-PCI FFR, without a significant effect on coronary microcirculation overall. Nevertheless, IMR increased in a group of patients and larger studies are warranted to explore predictors of microcirculatory response to postdilatation.

Turning the Mre11/Rad50 DNA repair complex on its head: lessons from SMC protein hinges, dynamic coiled-coil movements and DNA loop-extrusion?

The bacterial SbcC/SbcD DNA repair proteins were identified over a quarter of a century ago. Following the subsequent identification of the homologous Mre11/Rad50 complex in the eukaryotes and archaea, it has become clear that this conserved chromosomal processing machinery is central to DNA repair pathways and the maintenance of genomic stability in all forms of life. A number of experimental studies have explored this intriguing genome surveillance machinery, yielding significant insights and providing conceptual advances towards our understanding of how this complex operates to mediate DNA repair. However, the inherent complexity and dynamic nature of this chromosome-manipulating machinery continue to obfuscate experimental interrogations, and details regarding the precise mechanisms that underpin the critical repair events remain unanswered. This review will summarize our current understanding of the dramatic structural changes that occur in Mre11/Rad50 complex to mediate chromosomal tethering and accomplish the associated DNA processing events. In addition, undetermined mechanistic aspects of the DNA enzymatic pathways driven by this vital yet enigmatic chromosomal surveillance and repair apparatus will be discussed. In particular, novel and putative models of DNA damage recognition will be considered and comparisons will be made between the modes of action of the Rad50 protein and other related ATPases of the overarching SMC superfamily.

Contemporary management of stent thrombosis: Predictors of mortality and the role of new-generation drug-eluting stents.

OBJECTIVES: We sought to evaluate mortality predictors and the role of new-generation drug-eluting stents (NG-DES) in stent thrombosis (ST) management. BACKGROUND: No data are available regarding the outcome of patients with ST after interventional management that includes exclusively NG-DES. METHODS: Patients with definite ST of DES or BMS who underwent urgent/emergent angiography between 2015 and 2018 at our institution were considered for the study. After excluding patients who achieved TIMI-flow<2 after intervention or received an old-generation stent, 131 patients were included. Management classification was stent or non-stent treatment (medical management, thromboaspiration, balloon-angioplasty). Follow-up was performed to document all-cause death (ACD) and target-lesion-revascularization (TLR) that was used for censorship. RESULTS: Mode of presentation was STEMI in 88% and UA/NSTEMI in 12%. Type of ST was early, late, and very late in 11, 4, and 85%, respectively. Eighty four patients received stent and 47 non-stent treatment. After 926 ± 34 days, 21 ACDs, 7 TLRs and no cases of definite, recurrent ST were observed. Univariate predictors of in-hospital mortality were LVEF and presentation with shock or cardiac arrest. For patients discharged alive, non-stent treatment (HR 4.2, p = .01), TIMI-2 flow (HR 7.4, p = .002) and GFR < 60 mL/min (HR 3.8, p = .01) were independent predictors of ACD. The stent-treatment group had significantly better ACD-free survival after discharge, both unadjusted (p = .022) and adjusted (p = .018). CONCLUSIONS: After ST management, different predictors were observed for in-hospital mortality and mortality in patients discharged alive. The better outcome with NG-DES treatment is a novel observation, warranting further studies to elucidate if it is associated with stent-related or patient-related factors.

LC-ESI-QTOF-MS/MS Characterization of Seaweed Phenolics and Their Antioxidant Potential.

Seaweed is an important food widely consumed in Asian countries. Seaweed has a diverse array of bioactive compounds, including dietary fiber, carbohydrate, protein, fatty acid, minerals and polyphenols, which contribute to the health benefits and commercial value of seaweed. Nevertheless, detailed information on polyphenol content in seaweeds is still limited. Therefore, the present work aimed to investigate the phenolic compounds present in eight seaweeds [Chlorophyta (green), Ulva sp., Caulerpa sp. and Codium sp.; Rhodophyta (red), Dasya sp., Grateloupia sp. and Centroceras sp.; Ochrophyta (brown), Ecklonia sp., Sargassum sp.], using liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS/MS). The total phenolic content (TPC), total flavonoid content (TFC) and total tannin content (TTC) were determined. The antioxidant potential of seaweed was assessed using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay, a 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) free radical scavenging assay and a ferric reducing antioxidant power (FRAP) assay. Brown seaweed species showed the highest total polyphenol content, which correlated with the highest antioxidant potential. The LC-ESI-QTOF-MS/MS tentatively identified a total of 54 phenolic compounds present in the eight seaweeds. The largest number of phenolic compounds were present in Centroceras sp. followed by Ecklonia sp. and Caulerpa sp. Using high-performance liquid chromatography-photodiode array (HPLC-PDA) quantification, the most abundant phenolic compound was p-hydroxybenzoic acid, present in Ulva sp. at 846.083 ± 0.02 µg/g fresh weight. The results obtained indicate the importance of seaweed as a promising source of polyphenols with antioxidant properties, consistent with the health potential of seaweed in food, pharmaceutical and nutraceutical applications.

Evaluation of cytology and histopathology for the diagnosis of feline orbital neoplasia: 81 cases (2004-2019) and review of the literature.

OBJECTIVE: To provide an updated overview of feline orbital neoplasia, to compare diagnostic utility of cytology and histopathology, and to evaluate minimally invasive sampling modalities. PROCEDURES: A medical records search was performed to identify cats with orbital neoplasia. Data were collected regarding signalment, diagnosis, vision status, imaging modalities, and sample collection methods. A reference population with orbital neoplasia was also identified via literature search for comparison with regard to final diagnosis. RESULTS: Eighty-one cats met selection criteria and 140 cases were identified in the literature. In the study and reference populations, respectively, diagnoses were grouped as follows: round cell tumors 47% and 24%, epithelial tumors 38% and 40%, mesenchymal tumors 14% and 34%, and neurologic origin tumors 1% and 2%. The most common diagnoses in both groups were lymphoma and squamous cell carcinoma (SCC). Feline restrictive orbital myofibroblastic sarcoma (FROMS) was common in the reference population but not diagnosed in the study population. Cytology results were available for 41 cats; histopathology results were available for 65 cats. Both cytology and histopathology results were available for 25 cats, in 44% of which cytologic results were overturned. No significant complications were associated with any sampling method. Lack of cats with multiple samples available for histopathology limited comparison between tissue sampling methods. CONCLUSIONS: Orbital neoplasia is common in cats, with round cell and epithelial tumors diagnosed most commonly in the study population. Histopathology is superior to cytology in providing a definitive diagnosis. Minimally invasive tissue biopsy techniques appear to be safe and effective.

Evaluation of cytology and histopathology for the diagnosis of canine orbital neoplasia: 112 cases (2004-2019) and review of the literature.

OBJECTIVE: To provide an updated overview of canine orbital neoplasia, to compare diagnostic utility of cytology and histopathology, and to evaluate alternative sampling modalities, particularly image-guided core needle biopsy. PROCEDURES: A medical records search was performed to identify dogs with orbital neoplasia. Data were collected regarding signalment, diagnosis, vision status, imaging modalities, and sample collection methods. A reference population with orbital neoplasia was also identified via literature search for comparison with regard to final diagnosis. RESULTS: One hundred and twelve dogs met selection criteria. In the study and reference populations, respectively, diagnoses were grouped as follows: mesenchymal tumors 40% and 35%, epithelial tumors 35% and 18%, tumors of neural origin 8% and 37%, and round cell 17% and 10%. The most common diagnoses in the study group were nasal adenocarcinoma, osteosarcoma, lymphoma, and meningioma. Cytology results were available for 47 dogs and histopathology results were available for 95 dogs. Both cytology and histopathology results were available for 30 dogs, in 53% of which results were discordant. Cytology samples were nondiagnostic or provided a diagnosis that was later overturned in 32% of cases in which they were obtained. Results from core needle biopsy samples were nondiagnostic or overturned by surgical biopsy results in only 13% of cases. No significant complications were associated with any sampling method. CONCLUSIONS: Orbital neoplasia is common in dogs. Histopathology is superior to cytology in providing a definitive diagnosis. Image-guided core needle biopsy appears to be a safe and effective means of obtaining samples.

QTLs Associated with Resistance to Cardiomyopathy Syndrome in Atlantic Salmon.

Cardiomyopathy syndrome (CMS) caused by piscine myocarditis virus is a major disease affecting the Norwegian Atlantic salmon industry. Three different populations of Atlantic salmon from the Mowi breeding program were used in this study. The first 2 populations (population 1 and 2) were naturally infected in a field outbreak, while the third population (population 3) went through a controlled challenged test. The aim of the study was to estimate the heritability, the genetic correlation between populations and perform genome-wide association analysis for resistance to this disease. Survival data from population 1 and 2 and heart atrium histology score data from population 3 was analyzed. A total of 571, 4312, and 901 fish from population 1, 2, and 3, respectively were genotyped with a noncommercial 55,735 Affymetrix marker panel. Genomic heritability ranged from 0.12 to 0.46 and the highest estimate was obtained from the challenge test dataset. The genetic correlation between populations was moderate (0.51-0.61). Two chromosomal regions (SSA27 and SSA12) contained single nucleotide polymorphisms associated with resistance to CMS. The highest association signal (P = 6.9751 × 10-27) was found on chromosome 27. Four genes with functional roles affecting viral resistance (magi1, pi4kb, bnip2, and ha1f) were found to map closely to the identified quantitative trait loci (QTLs). In conclusion, genetic variation for resistance to CMS was observed in all 3 populations. Two important quantitative trait loci were detected which together explain half of the total genetic variance, suggesting strong potential application for marker-assisted selection and genomic predictions to improve CMS resistance.

Training the salmon's genes: influence of aerobic exercise, swimming performance and selection on gene expression in Atlantic salmon.

BACKGROUND: Farmed and wild Atlantic salmon are exposed to many infectious and non-infectious challenges that can cause mortality when they enter the sea. Exercise before transfer promotes growth, health and survival in the sea. Swimming performance in juveniles at the freshwater parr stage is positively associated with resistance to some diseases. Genetic variation is likely to affect response to exercise. In this study we map genetic differences associated with aerobic exercise, swimming performance and genetic origin. Eggs from the selectively bred Bolaks salmon and wild Lærdal River salmon strains were reared until parr in a common environment. Swimming performance was assessed by subjecting the fish to either continuous hard exercise or control conditions for 18 days. Heart was sampled for examination of gene expression using RNA-seq (~60 fish/treatment). RESULTS: Lower expression of genes affecting immune function was found in domesticated than wild parr. Among wild parr under control exercise the expression of a large number of genes involved in general metabolism, stress and immune response was lower in superior swimmers suggesting that minimisation of energy expenditure during periods of low activity makes parr better able to sustain bursts of swimming for predator avoidance. A similar set of genes were down-regulated with training among wild parr with inferior swimming performance. These parr react to training in a way that their cardiac expression patterns become like the superior performing wild parr under control exercise conditions. Diversifying selection caused by breeding of domesticated stock, and adaptive pressures in wild stock, has affected the expression and frequency of single nucleotide polymorphisms (SNPs) for multiple functional groups of genes affecting diverse processes. SNPs associated with swimming performance in wild parr map to genes involved in energetic processes, coding for contractile filaments in the muscle and controlling cell proliferation. CONCLUSIONS: Domesticated parr have less phenotypic plasticity in response to training and lower expression of genes with functions affecting immune response. The genetic response to training is complex and depends on the background of parr and their swimming ability. Exercise should be tailored to the genetics and swimming performance of fish.

The Hancock® Valved Conduit for Right Ventricular Outflow Tract Reconstruction in Sheep for Assessing New Devices.

BACKGROUND AND AIM OF THE STUDY: Xenograft conduits have been used successfully to repair congenital heart defects, but are prone to failure over time. Hence, in order to improve patient outcomes, better xenografts are being developed. When evaluating a conduit's performance and safety it must first be compared against a clinically available control in a large animal model. The study aim was to evaluate a clinically available xenograft conduit used in right ventricular outflow tract (RVOT) reconstruction in a sheep model. METHODS: RVOT reconstruction was performed in 13 adult and juvenile sheep, using the Medtronic Hancock® Bioprosthetic Valved Conduit (Hancock conduit). The method had previously been used on patients, and a newly modified variant termed 'RVOT Extraction' was employed to facilitate the surgical procedure. Animals were monitored over predetermined terms of 70 to 140 days. Serial transthoracic echocardiography, intracardiac pressure measurements and angiography were performed. On study completion the animals were euthanized and necropsies performed. RESULTS: Two animals died prior to their designated study term due to severe valvular stenosis and distal conduit narrowing, respectively. Thus, 11 animals survived the study term, with few or no complications. Generally, maximal and mean transvalvular pressure gradients across the implanted conduits were increased throughout the postoperative course. Among 11 full-term animals, seven conduits were patent with mild or no pseudointimal proliferation and with flexible leaflets maintaining the hemodynamic integrity of the valve. CONCLUSIONS: RVOT reconstruction using the Hancock conduit was shown to be successful in sheep, with durable and efficient performances. With its extensive clinical use in patients, and ability for long-term use in sheep (as described in the present study) it can be concluded that the Hancock conduit is an excellent control device for the evaluation of new xenografts in future preclinical studies.

A linkage map of transcribed single nucleotide polymorphisms in rohu (Labeo rohita) and QTL associated with resistance to Aeromonas hydrophila.

BACKGROUND: Production of carp dominates world aquaculture. More than 1.1 million tonnes of rohu carp, Labeo rohita (Hamilton), were produced in 2010. Aeromonas hydrophila is a bacterial pathogen causing aeromoniasis in rohu, and is a major problem for carp production worldwide. There is a need to better understand the genetic mechanisms affecting resistance to this disease, and to develop tools that can be used with selective breeding to improve resistance. Here we use a 6 K SNP array to genotype 21 full-sibling families of L. rohita that were experimentally challenged intra-peritoneally with a virulent strain of A. hydrophila to scan the genome for quantitative trait loci associated with disease resistance. RESULTS: In all, 3193 SNPs were found to be informative and were used to create a linkage map and to scan for QTL affecting resistance to A. hydrophila. The linkage map consisted of 25 linkage groups, corresponding to the number of haploid chromosomes in L. rohita. Male and female linkage maps were similar in terms of order, coverage (1384 and 1393 cM, respectively) and average interval distances (1.32 and 1.35 cM, respectively). Forty-one percent of the SNPs were annotated with gene identity using BLAST (cut off E-score of 0.001). Twenty-one SNPs mapping to ten linkage groups showed significant associations with the traits hours of survival and dead or alive (P <0.05 after Bonferroni correction). Of the SNPs showing significant or suggestive associations with the traits, several were homologous to genes of known immune function or were in close linkage to such genes. Genes of interest included heat shock proteins (70, 60, 105 and "small heat shock proteins"), mucin (5b precursor and 2), lectin (receptor and CD22), tributyltin-binding protein, major histocompatibility loci (I and II), complement protein component c7-1, perforin 1, ubiquitin (ligase, factor e4b isoform 2 and conjugation enzyme e2 c), proteasome subunit, T-cell antigen receptor and lymphocyte specific protein tyrosine kinase. CONCLUSIONS: A panel of markers has been identified that will be validated for use with both genomic and marker-assisted selection to improve resistance of L. rohita to A. hydrophila.

QTL for white spot syndrome virus resistance and the sex-determining locus in the Indian black tiger shrimp (Penaeus monodon).

BACKGROUND: Shrimp culture is a fast growing aquaculture sector, but in recent years there has been a shift away from tiger shrimp Penaeus monodon to other species. This is largely due to the susceptibility of P. monodon to white spot syndrome virus disease (Whispovirus sp.) which has impacted production around the world. As female penaeid shrimp grow more rapidly than males, mono-sex production would be advantageous, however little is known about genes controlling or markers associated with sex determination in shrimp. In this study, a mapped set of 3959 transcribed single nucleotide polymorphisms were used to scan the P. monodon genome for loci associated with resistance to white-spot syndrome virus and sex in seven full-sibling tiger shrimp families challenged with white spot syndrome virus. RESULTS: Linkage groups 2, 3, 5, 6, 17, 18, 19, 22, 27 and 43 were found to contain quantitative trait loci significantly associated with hours of survival after white spot syndrome virus infection (P < 0.05 after Bonferroni correction). Nine QTL were significantly associated with hours of survival. Of the SNPs mapping to these and other regions with suggestive associations, many were found to occur in transcripts showing homology to genes with putative immune functions of interest, including genes affecting the action of the ubiquitin-proteasome pathway, lymphocyte-cell function, heat shock proteins, the TOLL pathway, protein kinase signal transduction pathways, mRNA binding proteins, lectins and genes affecting the development and differentiation of the immune system (eg. RUNT protein 1A). Several SNPs significantly associated with sex were mapped to linkage group 30, the strongest associations (P < 0.001 after Bonferroni correction) for 3 SNPs located in a 0.8 cM stretch between positions 43.5 and 44.3 cM where the feminisation gene (FEM-1, affecting sexual differentiation in Caenorhabditis elegans) mapped. CONCLUSIONS: The markers for disease resistance and sexual differentiation identified by this study could be useful for marker assisted selection to improve resistance to WSSV and for identifying homogametic female individuals for mono-sex (all female) production. The genes with putative functions affecting immunity and sexual differentiation that were found to closely map to these loci provide leads about the mechanisms affecting these important economic traits in shrimp.

Analysis of immune-related ESTs and differential expression analysis of few important genes in lines of rohu (Labeo rohita) selected for resistance and susceptibility to Aeromonas hydrophila infection.

A total of 137,629 contigs generated via de novo transcriptome assembly from resistant and susceptible lines of rohu (first generation) raised against aeromoniasis were further analyzed in terms of defence-related genes. Out of 1,939 contigs showing homology to genes involved in immune processes, 1,866 were further categorised into different functional subgroups. Comparative analysis revealed five genes for the first time in any carp species out of which apolipoprotein h, septin 4 isoform 3 and septin isoform cra_c were identified for the first time in fish. Differential expression analysis of ten genes viz., heat shock proteins (Hsps) (Hsp30, Hsp70 and Hsp90), serum lectin isoform 1 (SLI1), linker histone H1M (LHH1M), NAD(P)H quinone 1 (NQO1), zona pellucida 2 (ZP2) and three unknown genes that were highly up-expressed in first generation resistant line fish from mRNA-seq coverage data, was carried out using susceptible and resistant individuals of the second generation selected populations in eight different tissues viz. liver, kidney, intestine, gill, brain, spleen, skin and muscle using qPCR. Significant up-regulation in Hsp90, NQO1, C_116914 and C_22454 in specific tissues of resistant line and variable expression in Hsp30 and LHH1M genes in different tissues of both lines were noticed. The expression of Hsp70 was lower in many tissues of the resistant line than in susceptible line rohu. The expression of ZP2, SLI1 and C_94589 genes was not significantly different in terms of fold difference between the two lines. Differentially expressed genes need further characterisation to explore their role in resistance to Aeromonas hydrophila infection in rohu.

Cryopreservation of sperm in farmed Australian greenlip abalone Haliotis laevigata.

This study investigated factors important to the development of the liquid nitrogen (LN) vapor sperm cryopreservation technique in farmed greenlip abalone Haliotis laevigata, including (1) cryoprotectant agent (CPA) toxicity; (2) cooling temperature (height above LN surface); (3) thawing temperature; (4) sperm to egg ratio; and (5) sugar supplementation, using sperm motility, fertilization rate or integrity/potential of sperm components and organelles as quality assessment indicators. Results suggested that among the single CPAs evaluated 6% dimethyl sulfoxide (Me2SO) would be the most suitable for sperm cryopreservation in this species. The highest post-thaw sperm motility was achieved with the sperm that had been exposed to LN vapor for 10min at 5.2cm above the LN surface, thawed and recovered in 60 and 18°C seawater bathes, respectively after at least 2h storage in LN. The highest fertilization rates were achieved at a sperm to egg ratio of 10,000:1 or 15,000:1. Addition of 1% glucose or 2% sucrose produced significantly higher post-thaw sperm motility than 6% Me2SO alone. Among the three cryoprotectant solutions further trialled, 6% Me2SO+1% glucose produced the highest fertilization rate of 83.6±3.7%. Evaluation of sperm has shown that the addition of glucose could significantly improve the sperm plasma membrane integrity and mitochondrial membrane potential. These results demonstrated a positive role of glucose in the improvement of sperm cryopreservation in farmed greenlip abalone.

Structural and functional insights into DNA-end processing by the archaeal HerA helicase-NurA nuclease complex.

Helicase-nuclease systems dedicated to DNA end resection in preparation for homologous recombination (HR) are present in all kingdoms of life. In thermophilic archaea, the HerA helicase and NurA nuclease cooperate with the highly conserved Mre11 and Rad50 proteins during HR-dependent DNA repair. Here we show that HerA and NurA must interact in a complex with specific subunit stoichiometry to process DNA ends efficiently. We determine crystallographically that NurA folds in a toroidal dimer of intertwined RNaseH-like domains. The central channel of the NurA dimer is too narrow for double-stranded DNA but appears well suited to accommodate one or two strands of an unwound duplex. We map a critical interface of the complex to an exposed hydrophobic epitope of NurA abutting the active site. Based upon the presented evidence, we propose alternative mechanisms of DNA end processing by the HerA-NurA complex.

End-resection at DNA double-strand breaks in the three domains of life.

During DNA repair by HR (homologous recombination), the ends of a DNA DSB (double-strand break) must be resected to generate single-stranded tails, which are required for strand invasion and exchange with homologous chromosomes. This 5'-3' end-resection of the DNA duplex is an essential process, conserved across all three domains of life: the bacteria, eukaryota and archaea. In the present review, we examine the numerous and redundant helicase and nuclease systems that function as the enzymatic analogues for this crucial process in the three major phylogenetic divisions.

Characterization of the ceruloplasmin gene and its potential role as an indirect marker for selection to Aeromonas hydrophila resistance in rohu, Labeo rohita.

Ceruloplasmin is an acute phase protein found to be activated by the host immune system during stress conditions. The ceruloplasmin gene has been reported in several teleosts and here we characterize the gene and test its association with resistance to Aeromonas hydrophila in rohu, Labeo rohita. A ceruloplasmin mRNA sequence of 3355 base pairs (bp) was derived (GenBank ID: JX010736). The coding sequence (CDS) comprised of 3276 bp that coded for 1092 amino acids. Alignment results showed the greatest similarity with zebrafish followed by channel catfish sequence, and a phylogenetic tree constructed on the basis of amino acid sequences showed that rohu shares a common clade with these two species. In the ontogeny study, the expression of ceruloplasmin was detected at 9 h post-fertilization onwards, and a strong level of expression was detected at 24 h (38-fold) and 15 days (34-fold) post-fertilization. The ceruloplasmin transcripts were evident in liver, spleen, stomach and heart. Expression was undetectable in gill, brain, eye, skin, muscle, intestine, anterior and posterior kidney tissues. Expression of ceruloplasmin after A. hydrophila infection was up-regulated 6 h post-challenge and was modulated until 15 days post-challenge. The level of ceruloplasmin was also compared in rohu selectively bred for higher growth and disease resistance. The gene showed a 4.58-fold higher level of expression in resistant line over susceptible line rohu selected based on family challenge test survival to A. hydrophila. Serum ceruloplasmin levels in three year classes of rohu selected for higher growth showed a positive correlation (0.49 ± 1.11) with survival against challenge with A. hydrophila. The estimated heritability was also found to be quite high (0.50 ± 0.22) for this parameter. Thus, ceruloplasmin could be one of the useful marker traits for selection against A. hydrophila resistance in fish.

Evolutionary predictions for a parasite metapopulation: Modelling salmon louse resistance to pest controls in aquaculture.

Pests often evolve resistance to pest controls used in agriculture and aquaculture. The rate of pest adaptation is influenced by the type of control, the selective pressure it imposes, and the gene flow between farms. By understanding how these factors influence evolution at the metapopulation level, pest management strategies that prevent resistance from evolving can be developed. We developed a model for the metapopulation and evolutionary dynamics of the salmon louse (Lepeophtheirus salmonis), which is a major parasite affecting salmon aquaculture. Different management scenarios were simulated across a network of salmon farms covering half of Norway, and their effects on louse epidemiology and evolution were investigated. We compared louse controls that differed in how they were deployed through time (discrete vs. continuous), how they impacted the louse life cycle, and in their overall efficacy. We adjusted the strength of selection imposed by treatments, the dominance effect of the resistant allele, and the geographic location at which resistance originated. Continuously acting strategies (e.g., louse-resistant salmon) were generally more effective than discrete strategies at controlling lice, especially when they increased louse mortality during early developmental stages. However, effective strategies also risked imposing frequent and/or strong selection on lice, thus driving rapid adaptation. Resistant alleles were more likely to be lost through genetic drift when they were recessive, had a low-fitness advantage, or originated in low-farm-density areas. The north-flowing current along the Norwegian coastline dispersed resistant genes from south to north, and limited gene flow in the opposite direction. We demonstrate how evolutionary models can produce quantitative predictions over large spatial and temporal scales and for a range of pest control scenarios. Quantitative outputs can be translated into practical management decisions applied at a regional level to minimise the risk of resistance developing.

Using cultured canine cardiac slices to model the autophagic flux with doxorubicin.

Chemotherapy-induced impairment of autophagy is implicated in cardiac toxicity induced by anti-cancer drugs. Imperfect translation from rodent models and lack of in vitro models of toxicity has limited investigation of autophagic flux dysregulation, preventing design of novel cardioprotective strategies based on autophagy control. Development of an adult heart tissue culture technique from a translational model will improve investigation of cardiac toxicity. We aimed to optimize a canine cardiac slice culture system for exploration of cancer therapy impact on intact cardiac tissue, creating a translatable model that maintains autophagy in culture and is amenable to autophagy modulation. Canine cardiac tissue slices (350 µm) were generated from left ventricular free wall collected from euthanized client-owned dogs (n = 7) free of cardiovascular disease at the Foster Hospital for Small Animals at Tufts University. Cell viability and apoptosis were quantified with MTT assay and TUNEL staining. Cardiac slices were challenged with doxorubicin and an autophagy activator (rapamycin) or inhibitor (chloroquine). Autophagic flux components (LC3, p62) were quantified by western blot. Cardiac slices retained high cell viability for >7 days in culture and basal levels of autophagic markers remained unchanged. Doxorubicin treatment resulted in perturbation of the autophagic flux and cell death, while rapamycin co-treatment restored normal autophagic flux and maintained cell survival. We developed an adult canine cardiac slice culture system appropriate for studying the effects of autophagic flux that may be applicable to drug toxicity evaluations.