From the Cover: Quantifying ecological, morphological, and genetic variation to delimit species in the coast horned lizard species complex (Phrynosoma)

Lineage separation and divergence form a temporally extended process whereby populations may diverge genetically, morphologically, or ecologically, and these contingent properties of species provide the operational criteria necessary for species delimitation. We inferred the historical process of lineage formation in the coast horned lizard (Phrynosoma coronatum) species complex by evaluating a diversity of operational species criteria, including divergence in mtDNA (98 specimens; 2,781 bp) and nuclear loci (RAG−1, 1,054 bp; BDNF 529 bp), ecological niches (11 bioclimatic variables; 285 unique localities), and cranial horn shapes (493 specimens; 16 landmarks). A phylogenetic analysis of mtDNA recovers 5 phylogeographic groups arranged latitudinally along the Baja California Peninsula and in California. The 2 southern phylogeographic groups exhibit concordance between genetic, morphological, and ecological divergence; however, differentiation is weak or absent at more recent levels defined by phylogeographic breaks in California. Interpreting these operational species criteria together suggests that there are 3 ecologically divergent and morphologically diagnosable species within the P. coronatum complex. Our 3-species taxonomic hypothesis invokes a deep coalescence event when fitting the mtDNA genealogy into the species tree, which is not unexpected for populations that have diverged recently. Although the hypothesis that the 3 phylogeographic groups distributed across California each represent distinctive species is not supported by all of the operational species criteria evaluated in this study, the conservation status of the imperiled populations represented by these genealogical units remains critical.     

Extensive and widespread homologies between mitochondrial DNA and chloroplast DNA in plants

We used hybridization techniques to demonstrate that numerous sequence homologies exist between cloned mung bean and spinach chloroplast DNA (ctDNA) restriction fragments and mtDNAs from corn, mung bean, spinach, and pea. The strongest cross-homologies are between clones derived from the ctDNA inverted repeat and mtDNA from corn and pea, although all the ctDNA clones tested hybridized to at least one mtDNA restriction fragment. Known chloroplast genes showing strong mtDNA homologies include those for the large subunit of ribulosebisphosphate carboxylase, which hybridizes to corn mtDNA, and the β subunit of the chloroplast ATPase, which hybridizes to mung bean mtDNA. Certain of these homologies were confirmed by using cloned spinach mtDNA restriction fragments as probes in reciprocal hybridizations to ctDNA. Several of these ctDNA-homologous mtDNA sequences were shown to be much more closely related to ctDNA from the same species than to that of a distantly related species. We interpret these differential homologies as evidence for relatively recent DNA sequence transfer events, suggesting that transpostion between the two genomes is an ongoing evolutionary process.     

Phylogeography of Androctonus species (Scorpiones: Buthidae) in Tunisia: Diagnostic characters for linking species to scorpionism

A fragment of the mitochondrial (mt) 16S ribosomal RNA gene was amplified by PCR and sequenced from individual adult scorpions of the genus Androctonus, which were sampled from central and southern Tunisia and identified using an explicit set of morphological characters. Phylogenetic analyses placed the mtDNA haplotypes in three well-supported monophyletic lineages, corresponding to the morphospecies Androctonus aeneas, Androctonus amoreuxi and Androctonus australis. The latter species was the most abundant and widespread, and it was characterized by two mtDNA sub-lineages each of which predominated only north or south of the Chott el Jerid, a seasonally flooded saline depression that divides non-Mediterranean Tunisia. The divergence of the two mtDNA lineages was dated by mtDNA molecular clocks, indicating that the formation of the Chott el Jerid is unlikely to have been the barrier generating the vicariant evolution of the two lineages of A. australis, although it may have impeded their mixing following secondary contact. Both regional mtDNA lineages were found in A. australis hector and A. australis garzonii, indicating that these two morphological forms are neither monophyletic nor geographically isolated and, therefore, should not be treated as species or subspecies. It is recommended that no subspecies of A. australis should be recognized in North Africa and toxicologists should cease the taxonomic error of referring to a species “Androctonus australis Hector”. The morphological form “hector” has no proven association with an increased risk of scorpionism compared with “garzonii”. However, it might be prudent to produce anti-venom in Tunisia by using both morphological forms of A. australis collected each side of the Chott el Jerid, because of the evidence for regional variation in toxins. The highest risk for scorpion stings occurs in the central region, where the new diagnostic markers should be used to discover any association between Androctonus species and scorpionism.     

Transmission of Grapevine leafroll-associated virus-1 (Ampelovirus) and Grapevine virus A (Vitivirus) by the Cottony Grape Scale,Pulvinaria vitis (Hemiptera: Coccidae)

The cottony grape scale Pulvinaria vitis is a scale insect colonizing grapevine; however, its capacity as a vector of grapevine viruses is poorly known in comparison to other scale species that are vectors of viral species in the genera Ampelovirus and Vitivirus. The ability of P. vitis to transmit the ampeloviruses Grapevine leafroll-associated viruses [GLRaV]−1, −3, and −4, and the vitivirus Grapevine virus A (GVA), to healthy vine cuttings was assessed. The scale insects used originated from commercial vine plots located in Alsace, Eastern France. When nymphs sampled from leafroll-infected vineyard plants were transferred onto healthy cuttings, only one event of transmission was obtained. However, when laboratory-reared, non-viruliferous nymphs were allowed to acquire viruses under controlled conditions, both first and second instar nymphs derived from two vineyards were able to transmit GLRaV−1 and GVA. This is the first report of GLRaV−1 and GVA transmission from grapevine to grapevine by this species.     

Isolation and characterization of nodulation genes from Bradyrhizobium sp. (Vigna) strain IRc 78

An 11.76-kilobase-pair (kb) segment of DNA from Bradyrhizobium sp. (Vigna) strain IRc 78 that hybridizes to nodulation genes of Rhizobium meliloti strain 41 was isolated. Hybridization of the 11.76-kb DNA fragment to DNA from other Bradyrhizobium species revealed a high degree of sequence conservation in this region. Transfer of the 11.76-kb segment to nodulation-defective (Nod^-) mutants of R. meliloti restored their ability to induce nodules on Medicago sativa (alfalfa). Mutants of strain IRc 78 generated by Tn5 mutagenesis of the 11.76-kb segment fell into three classes according to their symbiotic reaction with Vigna unguiculata (cowpea). Class I mutants of strain IRc 78 were unable to induce root-hair curling or to nodulate; class II induced small, ineffective nodules; and class III showed delayed and decreased nodulation with reduction in amount of nitrogen fixed. Furthermore, in contrast to the wild-type strain, class I mutants could not induce nodules on Glycine max (soybean), Cajanus cajan (pigeon pea), or Arachis hypogaea (peanut). This finding suggests a common function of the 11.76-kb region in the infection of host plants by Bradyrhizobium either through root hairs or by “crack entry.”     

Intrinsic karyotype stability and gene copy number variations may have laid the foundation for tetraploid wheat formation

Polyploidy or whole-genome duplication is recurrent in plant evolution, yet only a small fraction of whole-genome duplications has led to successful speciation. A major challenge in the establishment of nascent polyploids is sustained karyotype instability, which compromises fitness. The three putative diploid progenitors of bread wheat, with AA, SS (S ∼ B), and DD genomes occurred sympatrically, and their cross-fertilization in different combinations may have resulted in fertile allotetraploids with various genomic constitutions. However, only SSAA or closely related genome combinations have led to the speciation of tetraploid wheats like Triticum turgidum and Triticum timopheevii. We analyzed early generations of four newly synthesized allotetraploid wheats with genome compositions S^shS^shA^mA^m, S^lS^lAA, S^bS^bDD, and AADD by combined fluorescence and genomic in situ hybridization-based karyotyping. Results of karyotype analyses showed that although S^shS^shA^mA^m and S^lS^lAA are characterized by immediate and persistent karyotype stability, massive aneuploidy and extensive chromosome restructuring are associated with S^bS^bDD and AADD in which parental subgenomes showed markedly different propensities for chromosome gain/loss and rearrangements. Although compensating aneuploidy and reciprocal translocation between homeologs prevailed, reproductive fitness was substantially compromised due to chromosome instability. Strikingly, localized genomic changes in repetitive DNA and copy-number variations in gene homologs occurred in both chromosome stable lines, S^shS^shA^mA^m and S^lS^lAA. Our data demonstrated that immediate and persistent karyotype stability is intrinsic to newly formed allotetraploid wheat with genome combinations analogous to natural tetraploid wheats. This property, coupled with rapid gene copy-number variations, may have laid the foundation of tetraploid wheat establishment.Polyploidy or whole-genome duplication (WGD) is a driving force in plant and vertebrate evolution (1–5). However, recent molecular phylogenetic studies have argued against a general creative role of WGD in plant evolution (6–8). This incongruence in opinions is long standing; in fact Stebbins in his seminal work in 1970s stated, “polyploidy has contributed little to progressive evolution” (9). Clearly, the two schools of thought regarding the roles of WGD in plant evolution have existed for years, and can converge if we accept the idea that WGDs have occurred frequently in nature but only a small fraction thereof have contributed to successful speciation (7, 10). However, the genetic and genomic factors determining one of the two fundamental fates for a nascent polyploid remained elusive.The Triticum–Aegilops complex includes both diploid and polyploid species with phylogenetically well-defined organismal relationships (11). This plant group is therefore suitable to address the issue raised above. This is especially so because all polyploid species of the Triticum–Aegilops complex represent examples of evolutionarily recent successful speciation events via allopolyploidization, i.e., hybridization and doubling of genomes from Triticum/Aegilops species (11). Speciation of allohexaploid common wheat, Triticum aestivum L., the founder crop for the initial establishment of Middle-Eastern and European agriculture, is characterized by two sequential polyploidization events, i.e., allotetraploidization and allohexaploidization. The former event involved hybridization of two diploid species, Triticum urartu (genome AA) and a yet-unknown or extinct goat-grass species of the genus Aegilops section Sitopsis (genome SS ∼ BB). This event led to the speciation of allotetraploid emmer wheat, Triticum turgidum ssp. dicoccoides (12). The latter event that involved hybridization of a primitive domesticated form of T. turgidum (genome BBAA) with a goat-grass species Aegilops tauschii (genome DD) led to the speciation of common wheat (11). In parallel, allohexaploidization by hybridization of another allotetraploid wheat, Triticum timopheevii (genome GGAA) (13) and Einkorn wheat, Triticum monococcum (genome A^mA^m) has resulted in the speciation of Triticum zhukovskyi (genome GGAAA^mA^m) (14) (Fig. S1A). Thus, the natural hexaploid bread wheat has three diploid progenitors (13) (Fig. S1A) that have diverged from a common ancestor only about 2.5–4.5 Mya (15). Therefore, it is perhaps not surprising that allotetraploidization by hybridization of any two of these three diploid species can still be accomplished artificially to produce fertile tetraploid plants (16, 17) (Fig. S1B). Furthermore, the diploid progenitor species are known to exist sympatrically across several geographical areas in the Eastern Mediterranean region and the Near East (13). These features of the diploid progenitor species of polyploid wheat raise an intriguing question: Why has only the genome combination of SSAA or closely related ones led to successful speciation of the two natural allotetraploid wheat species, T. turgidum and T. timopheevii? This question is germane to the more general issue as to why only a small fraction of WGDs have led to successful speciation in the evolutionary history of angiosperms.A major challenge for the establishment of newly formed allopolyploids as new species is sustained karyotype instability, in particular, whole-chromosome aneuploidy, which, at the organismal level, is associated with compromised cellular metabolism and reduced fitness (18). In newly formed allohexaploid wheat, extensive whole-chromosome aneuploidy was reported (19, 20). However, the immediate chromosomal consequences associated with formation of allotetraploid wheat have remained uninvestigated.Here, we conducted in-depth molecular cytogenetic analyses of karyotype stability using fluorescence and genomic in situ hybridization (FISH and GISH) techniques and performed locus-specific molecular genetic analysis of gene copy-number variations (CNVs) in a set of protein-coding genes using newly synthesized allotetraploid wheats. Different genomic combinations of diploid Triticum/Aegilops species that are parsimoniously representing the diploid progenitors of tetraploid and hexaploid wheats were used in this study. We document that dramatic differences in karyotype stability of both chromosome number and structure existed between the S^shS^shA^mA^m/S^lS^lAA versus the S^bS^bDD/AADD genome combinations. Remarkably, localized loss or gain of DNA repeats and CNVs of gene homologs occurred widely in both chromosome stable lines, S^shS^shA^mA^m and S^lS^lAA, which, in their genomic constitutions, mimic natural tetraploid wheats, T. turgidum and T. timopheevii. We propose that persistent karyotype stability coupled with localized genomic changes and rapid CNVs of gene homologs, have laid the foundation for successful tetraploid wheat establishment and speciation.     

Targeted genotyping of circulating tumor DNA for classical Hodgkin lymphoma monitoring: a prospective study

The relevance of circulating tumor DNA (ctDNA) analysis as a liquid biopsy and minimal residual disease tool in the management of classical Hodgkin lymphoma (cHL) patients was demonstrated in retrospective settings and remains to be confirmed in a prospective setting. We developed a targeted Next-Generation sequencing (NGS) panel for fast analysis (AmpliSeq® technology) of nine commonly mutated genes in biopies and ctDNA of cHL patients. We then conducted a prospective trial to assess ctDNA follow-up at diagnosis and after two cycles (C2) of chemotherapy. Sixty cHL patients treated by first line conventional chemotherapy (BEACOPPescalated [21.3%], ABVD/ABVD-like [73.5%] and other regimens [5.2%, for elderly patients]) were assessed in this noninterventional study. The median age of the patients was 33.5 years (range: 20-86). Variants were identified in 42 (70%) patients. Mutations of NFKBIE, TNFAIP3, STAT6, PTPN1, B2M, XPO1, ITPKB, GNA13 and SOCS1 were found in 13.3%, 31.7%, 23.3%, 5%, 33.3%, 10%, 23.3%, 13.3% and 50% of patients, respectively. ctDNA concentration and genotype were correlated with clinical characteristics and presentation. Regarding early therapeutic response, 45 patients (83%, not available [NA] =6) had a negative positron emission tomography (PET) after C2 (Deauville Score 1-3). The mean of DeltaSUVmax after C2 was -78.8%. ctDNA after C2 was analysed in 54 patients (90%). ctDNA became rapidly undetectable in all cases after C2. Variant detection in ctDNA is suitable to depict the genetic features of cHL at diagnosis and may help to assess early treatment response, in association with PET. Clinical Trial reference: {"type":"clinical-trial","attrs":{"text":"NCT02815137","term_id":"NCT02815137"}}NCT02815137.     

Mitochondrial DNA deletions in dilated cardiomyopathy: A clinical study employing endomyocardial sampling

Objectives. The aim of this study was to assess the occurrence of the two most commonly encountered mitochondrial DNA (mtDNA) deletions in the hearts of patients with idiopathic dilated cardiomyopathy.Background. The mutation frequency of mtDNA is high, and sporadic cases of cardiomyopathies associated with mtDNA deletions have been described. Reports of increases in mtDNA deletions with advancing age also exist.Methods. We studied 15 consecutive patients with typical signs of idiopathic dilated cardiomyopathy, without a family history, together with 16 control hearts obtained at autopsy from patients who died of noncardiac causes. The patients underwent both right and left heart catheterization, during which endomyocardial biopsy samples were taken. The mtDNA in these samples and in the control hearts was analyzed by the polymerase chain reaction technique for the occurrence and proportion of 5- and 7.4-kilobase (kb) deletions (Cambridge sequence map positions from nucleotides 8469 to 13447 and 8637 to 16084, respectively).Results. The 5-kb mtDNA deletion was observed in the hearts of all of the patients with idiopathic dilated cardiomyopathy, accounting for 0.32 ± 0.05% (mean ± SEM) of the total mtDNA. The 7.4-kb deletion was found in 7 of the 15 patients with idiopathic dilated cardiomyopathy and comprised 0.28 ± 0.08% of the total. The 5- and 7.4-kb deletions were detected in 12 and 9 control hearts, respectively, quantitatively similar to the patients with idiopathic dilated cardiomyopathy. A sigmoidal age dependency of the mtDNA deletions was found both in the patients with cardiomyopathy and in the control hearts, but after elimination of the confounding age variable, there was no difference between these groups.Conclusions. Because of the similarity of the age-dependent increase in the frequency of mtDNA deletions in cardiomyopathic and control hearts, the deletions have no causal relation with idiopathic dilated cardiomyopathy. The present results confirm the notion of an increase in mtDNA deletions with advancing age and show that endomyocardial tissue sampling is a feasible method for detecting mtDNA defects in affected hearts.     

Molecular archaeology of the Escherichia coli genome

The availability of the complete sequence of Escherichia coli strain MG1655 provides the first opportunity to assess the overall impact of horizontal genetic transfer on the evolution of bacterial genomes. We found that 755 of 4,288 ORFs (547.8 kb) have been introduced into the E. coli genome in at least 234 lateral transfer events since this species diverged from the Salmonella lineage 100 million years (Myr) ago. The average age of introduced genes was 14.4 Myr, yielding a rate of transfer 16 kb/Myr/lineage since divergence. Although most of the acquired genes subsequently were deleted, the sequences that have persisted (≈18% of the current chromosome) have conferred properties permitting E. coli to explore otherwise unreachable ecological niches.     

Limited oral bioavailability and active epithelial excretion of paclitaxel (Taxol) caused by P-glycoprotein in the intestine

In mice, the mdr1a and mdr1b genes encode drug-transporting proteins that can cause multidrug resistance in tumor cells by lowering intracellular drug levels. These P-glycoproteins are also found in various normal tissues such as the intestine. Because mdr1b P-glycoprotein is not detectable in the intestine, mice with a homozygously disrupted mdr1a gene [mdr1a(−/−) mice] do not contain functional P-glycoprotein in this organ. We have used these mdr1a(−/−) mice to study the effect of gut P-glycoprotein on the pharmacokinetics of paclitaxel. The area under the plasma concentration-time curves was 2- and 6-fold higher in mdr1a(−/−) mice than in wild-type (wt) mice after i.v. and oral drug administration, respectively. Consequently, the oral bioavailability in mice receiving 10 mg paclitaxel per kg body weight increased from only 11% in wt mice to 35% in mdr1a(−/−) mice. The cumulative fecal excretion (0–96 hr) was markedly reduced from 40% (after i.v. administration) and 87% (after oral administration) of the administered dose in wt mice to below 3% in mdr1a(−/−) mice. Biliary excretion was not significantly different in wt and mdr1a(−/−) mice. Interestingly, after i.v. drug administration of paclitaxel (10 mg/kg) to mice with a cannulated gall bladder, 11% of the dose was recovered within 90 min in the intestinal contents of wt mice vs. <3% in mdr1a(−/−) mice. We conclude that P-glycoprotein limits the oral uptake of paclitaxel and mediates direct excretion of the drug from the systemic circulation into the intestinal lumen.     

Isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint (Mentha x piperita, L.) that produces the aphid alarm pheromone (E)-β-farnesene

(E)-β-Farnesene is a sesquiterpene semiochemical that is used extensively by both plants and insects for communication. This acyclic olefin is found in the essential oil of peppermint (Mentha x piperita) and can be synthesized from farnesyl diphosphate by a cell-free extract of peppermint secretory gland cells. A cDNA from peppermint encoding (E)-β-farnesene synthase was cloned by random sequencing of an oil gland library and was expressed in Escherichia coli. The corresponding synthase has a deduced size of 63.8 kDa and requires a divalent cation for catalysis (K_m for Mg²⁺ ≈ 150 μM; K_m for Mn²⁺ ≈ 7 μM). The sesquiterpenoids produced by the recombinant enzyme, as determined by radio-GC and GC-MS, are (E)-β-farnesene (85%), (Z)-β-farnesene (8%), and δ-cadinene (5%) with the native C15 substrate farnesyl diphosphate (K_m ≈ 0.6 μM; V_rel = 100) and Mg²⁺ as cofactor, and (E)-β-farnesene (98%) and (Z)-β-farnesene (2%) with Mn²⁺ as cofactor (V_rel = 80). With the C₁₀ analog, GDP, as substrate (K_m = 1.5 μM; V_rel = 3 with Mg²⁺ as cofactor), the monoterpenes limonene (48%), terpinolene (15%), and myrcene (15%) are produced.     

Citrate 4% versus Heparin and the Reduction of Thrombosis Study (CHARTS)

Background and objectives: Citrate 4% has antithrombotic and antibacterial properties, which makes it a potentially superior alternative to heparin as an indwelling intraluminal locking agent.Design, setting, participants, and measurements: Sixty-one prevalent hemodialysis (HD) patients dialyzing with a tunneled cuffed HD catheter were randomized in a pilot study to receive either heparin 5000 U/ml or citrate 4% as a locking agent after HD. The primary outcomes were the development of catheter dysfunction (defined as a blood pump speed <250 ml/min or the use of tissue plasminogen activator) and catheter-associated bacteremia. The secondary outcomes were the development of an exit-site infection or bleeding complications (either local or systemic).Results: Citrate had comparable catheter dysfunction episodes to heparin (13/32 [41%] cases versus 12/29 [41%] cases, respectively). There were no differences in the development of catheter-associated bacteremia (2.2/1000 catheter days citrate versus 3.3/1000 catheter days heparin group; P = 0.607) or exit-site infection (2.2/1000 catheter days for both groups).Conclusions: The preliminary findings from our pilot study demonstrate that 4% citrate is effective in maintaining catheter patency and does not appear to have any increased incidence of infections. Because citrate is significantly cheaper and has a more favorable side effect profile than heparin, it can be considered a potentially better locking agent in HD catheters.Catheter use among hemodialysis (HD) patients continues to be high; in fact, recent data indicates that up to 33% of patients in Canada are dialyzing with a catheter (1). Complications of catheters are well known and include catheter dysfunction (CD), infection, and central vein stenosis. The burden of catheter-associated infections contributes to morbidity and subsequent mortality in HD patients. Catheter-related infections may start with bacterial colonization of the catheter hub or exit site and lead to subsequent exit-site infection (ESI) with or without bacteremia.The use of a catheter and all of its associated complications significantly increases the cost of care in these patients as compared with a native arteriovenous fistula (2). There is a renewed interest in citrate as an alternate to heparin as a locking solution in HD catheters because of its antithrombotic and antibacterial properties and the reduced costs relative to heparin. Furthermore, complications of heparin include local and systemic bleeding events as well as the potential for thrombocytopenia (3). Citrate may be a useful alternative to heparin because it is not known to produce the complications of thrombocytopenia or bleeding.Despite the use of citrate 4% in many HD units there is only one published randomized trial that compares citrate 4% and heparin in the HD catheter population (4). This study allocated 30 patients with temporary catheters to citrate 4%, heparin 5000 U/ml or polygeline (4). Unfortunately this study was not designed to compare outcomes of infection or thrombosis and the main outcome (i.e., visible clot formation in the catheter) is of questionable clinical relevance. Two prospective observational trials (5,6) recently examined the rate of catheter exchange, tissue plasminogen activator (TPA) use, and bacteremias in a HD population who were converted from heparin to citrate 4%. These studies gave conflicting results, with Lok et al. demonstrating significant reductions in catheter exchange rates, TPA use, and bacteremias in the citrate group whereas Grudzinski et al. found no reduction in catheter exchanges or bacteremias.Weijmer et al. performed a randomized trial involving 210 patients (98 tunneled cuffed catheters and 193 uncuffed catheters) who received either heparin 5000 U/ml or citrate 43% (7). There was a significant reduction in catheter-associated bacteremia (CAB): 1.1/1000 catheter days for citrate and 4.1 catheter days in the heparin group (P < 0.01) but no difference in the CD episodes.Initial studies of citrate were halted because of cardiac toxicity of 43% solutions (8); recent advances have demonstrated that 4% solutions are safe and effective, but direct comparisons of citrate to heparin are limited and have been performed in variable populations with different outcomes (9,10). We conducted a pilot study using a randomized design to compare the effect of citrate 4% and 5000 U/ml heparin in terms of CAB, ESI, and thrombotic episodes in a Canadian cohort of prevalent dialysis patients with cuffed catheters. The purpose of this pilot study is to assess the feasibility of pursuing a large, multicenter, quasi-randomized trial by exploring the resources and recruitment methods required.     

Circulating tumor DNA (ctDNA) to evaluate minimal residual disease (MRD), treatment response,and posttreatment prognosis in pancreatic adenocarcinoma

BackgroundCirculating tumor DNA (ctDNA) has emerged as a blood-based test with multiple utilities in oncology. In the past few years, multiple studies of varying designs, methods, and quality have emerged which show promise for ctDNA as a tool to assess response to treatment and detect minimal residual disease (MRD) across various gastrointestinal (GI) malignancies. We aim to review the current literature for ctDNA as it pertains to assessing treatment response, MRD, prognosis, and risk of recurrence for pancreatic adenocarcinoma.MethodsPubMed was queried with a combination of terms regarding pancreatic adenocarcinoma, minimal residual disease, resection, and prognosis. All resultant articles were reviewed by the authors for appropriate fit with scope.ResultsFourteen articles were identified that fit with the scope of this review.ConclusionsDetectable ctDNA after definitive resection, specifically mutated KRAS, correlates with shorter recurrence-free survival (RFS), overall survival (OS), and overall prognosis. Limited data also suggests ctDNA may provide a noninvasive means to assess response to chemotherapy. Whether this information is actionable in terms of altering neoadjuvant or postresection treatment regimens remains an open question requiring further study.     

Amyrel, a paralogous gene of the amylase gene family in Drosophila melanogaster and the Sophophora subgenus

We describe a gene from Drosophila melanogaster related to the alpha-amylase gene Amy. This gene, which exists as a single copy, was named Amyrel. It is strikingly divergent from Amy because the amino acid divergence is 40%. The coding sequence is interrupted by a short intron at position 655, which is unusual in amylase genes. Amyrel has also been cloned in Drosophila ananassae, Drosophila pseudoobscura, and Drosophila subobscura and is likely to be present throughout the Sophophora subgenus, but, to our knowledge, it has not been detected outside. Unexpectedly, there is a strong conservation of 5′ and 3′ flanking regions between Amyrel genes from different species, which is not the case for Amy and which suggests that selection acts on these regions. In contrast to the Amy genes, Amyrel is transcribed in larvae of D. melanogaster but not in adults. However, the protein has not been detected yet. Amyrel evolves about twice as fast as Amy in the several species studied. We suggest that this gene could result from a duplication of Amy followed by accelerated and selected divergence toward a new adaptation.     

Management of chronic heart failure in the community: role of a hospital based open access heart failure service

Objective: To evaluate the role of an open access heart failure service based at a teaching hospital for the diagnosis and treatment optimisation of patients with heart failure in the community and to identify measures that may further enhance the effectiveness of such a service.

Subjects: 963 patients with suspected heart failure seen over an eight year period referred by their general practitioners to the cardiology department at a district general hospital.

Main outcome measures: Presence or absence of left ventricular systolic dysfunction (LVSD) (left ventricular ejection fraction < 50% on echocardiography), and determination of the risk factors and predictors of LVSD.

Results: The majority of the patients were women (60% v 40%) and elderly (mean age 68.8 years). On echocardiography, only 30.8% were found to have LVSD. Patients were more likely to have LVSD if they were men (42.3% v 23.1%, p < 0.001, relative risk (RR) 1.8), were > 60 years of age (33.5% v 20.8%, p < 0.001, RR 1.6), or had a history of diabetes (49.4% v 29.1%, p < 0.001, RR 1.7), ischaemic heart disease (36.5% v 29.1%, p = 0.04, RR 1.3), or atrial fibrillation (52.6% v 27.8%, p < 0.001, RR 1.9). An abnormal ECG (48.4% v 19.5%, p < 0.001, RR 2.5) and cardiothoracic ratio > 0.5 on chest radiograph (44.3% v 17.8%, p < 0.001, RR 2.5) were found to be good predictors of LVSD. A normal ECG (negative predictive value 80.5%) and a cardiothoracic ratio of < 0.5 (negative predictive value 82.2%) can be used as baseline measures to identify patients with lower risk of developing LVSD (combined negative predictive value 87.9%).

Conclusions: An open access heart failure clinic is effective for the diagnosis and management of chronic heart failure in community based patients. The presence of risk factors and simple baseline tests can be used to identify patients with LVSD in the community. The introduction of a protocol based on these findings into a referral system can improve the efficiency and cost effectiveness of such a service.

     

Gene targeting by linear duplex DNA frequently occurs by assimilation of a single strand that is subject to preferential mismatch correction

To study targeted recombination, a single linear 2-kb fragment of LEU2 DNA was liberated from a chromosomal site within the nucleus of Saccharomyces cerevisiae, by expression of the site-specific HO endonuclease. Gene targeting was scored by gene conversion of a chromosomal leu2 mutant allele by the liberated LEU2 fragment. This occurred at a frequency of only 2 × 10⁻⁴, despite the fact that nearly all cells successfully repaired, by single-strand annealing, the chromosome break created by liberating the fragment. The frequency of Leu⁺ recombinants was 6- to 25-fold higher in pms1 strains lacking mismatch repair. In 70% of these cases, the colony was sectored for Leu⁺/Leu⁻. Similar results were obtained when a 4.1-kb fragment containing adjacent LEU2 and ADE1 genes was liberated, to convert adjacent leu2 and ade1 mutations on the chromosome. These results suggest that a linear fragment is not assimilated into the recipient chromosome by two crossovers each close to the end of the fragment; rather, heteroduplex DNA between the fragment and the chromosome is apparently formed over the entire region, by the assimilation of one of the two strands of the linear duplex DNA. Moreover, the recovery of Leu⁺ transformants is frequently defeated by the cell’s mismatch repair machinery; more than 85% of mismatches in heteroduplex DNA are corrected in favor of the resident, unbroken (mutant) strand.     

Effect of phosphorus supplementation on weight gain and waist circumference of overweight/obese adults: a randomized clinical trial

Background:

Phosphorus status is inversely correlated with body weight; however, the effect of phosphorus supplementation on body weight in a controlled design has not been studied.

Methods:

This is a double-blind, randomized, placebo-controlled trial of 63 adults aged 18–45 years with a body mass index (BMI) of ⩾25 kg m⁻² and normal kidney function at the American University of Beirut. Participants were randomly assigned to the placebo or phosphorus group where daily placebo or phosphorus supplements were ingested with three main meals (breakfast, lunch and dinner) for a period of 12 weeks. Primary outcomes were changes in anthropometric measures, blood metabolites (including lipid profile, glucose and insulin) and subjective appetite scores. The trial is registered with Clinical Trial.gov, {"type":"clinical-trial","attrs":{"text":"NCT02329990","term_id":"NCT02329990"}}NCT02329990.

Results:

Body weight was significantly lower in the phosphorus group when compared with the placebo group (−0.65 kg (95% confidence interval (CI) −1.69 to 0.40) vs 1.13 kg (95% CI 0.19 to 2.06), P=0.01). Similarly, BMI and waist circumference were significantly lower in the phosphorus group when compared with the placebo group (−0.24 kg m⁻² (95% CI −0.59 to 0.12) vs 0.42 kg m⁻² (95% CI 0.05 to 0.78), P=0.01; −3.62 cm (95% CI−4.90 to −2.33) vs 0.38 cm ( 95% CI−0.44 to 1.20), P<0.001; respectively). Several parameters of subjective appetite scores were decreased in the phosphorus-supplemented group.

Conclusions:

Phosphorus supplementation for 12 weeks significantly decreases body weight, BMI, waist circumference and subjective appetite scores. These findings support a promising role of the mineral phosphorus in the prevention and management of obesity, especially abdominal adiposity. The exact mechanisms of action and longer-term effects still need to be elucidated.     

The OPTIMA Study: Assessing a New Cinacalcet (Sensipar/Mimpara) Treatment Algorithm for Secondary Hyperparathyroidism

Background and objectives: Cinacalcet, a novel calcimimetic, targets the calcium-sensing receptor to lower parathyroid hormone (PTH), calcium, and phosphorus levels in dialysis patients with secondary hyperparathyroidism (SHPT). This study compared the efficacy of a cinacalcet-based regimen with unrestricted conventional care (vitamin D and phosphate binders) for achieving the stringent National Kidney Foundation Kidney Disease Outcomes Quality Initiative (KDOQI) targets for dialysis patients.Study design: In this multicenter, open-label study, hemodialysis patients with poorly controlled SHPT were randomized to receive conventional care (n = 184) or a cinacalcet-based regimen (n = 368). Doses of cinacalcet, vitamin D sterols, and phosphate binders were adjusted during a 16-wk dose-optimization phase with the use of algorithms that allowed cinacalcet to be used with adjusted doses of vitamin D. The primary end point was the proportion of patients with mean intact PTH ≤300 pg/ml during a 7-wk efficacy assessment phase.Results: A higher proportion of patients receiving the cinacalcet-based regimen versus conventional care achieved the targets for PTH (71% versus 22%, respectively; P < 0.001), Ca × P (77% versus 58%, respectively; P < 0.001), calcium (76% versus 33%, respectively; P < 0.001), phosphorus (63% versus 50%, respectively; P = 0.002), and PTH and Ca × P (59% versus 16%, respectively, P < 0.001), and allowed a 22% reduction in vitamin D dosage in patients receiving vitamin D at baseline. Achievement of targets was greatest in patients with less severe disease (intact PTH range, 300 to 500 pg/ml) and the cinacalcet dose required was lower in these patients (median = 30 mg/d).Conclusions: Compared with conventional therapy, a cinacalcet-based treatment algorithm increased achievement of KDOQI treatment targets in dialysis patients in whom conventional therapy was no longer effective in controlling this disease.Secondary hyperparathyroidism (SHPT), characterized by parathyroid hyperplasia and persistently elevated plasma levels of parathyroid hormone (PTH), commonly accompanies chronic kidney disease (CKD) (1–4). A major complication of SHPT is renal osteodystrophy, whereas alterations in calcium and phosphorus metabolism additionally contribute to soft tissue calcification (including cardiovascular calcification) (5–7). Elevated serum levels of PTH, phosphorus, and calcium × phosphorus ion product (Ca × P) are associated with an increased mortality risk (8–11). In recognition of this, the National Kidney Foundation Kidney Disease Outcome Quality Initiative (KDOQI) has published treatment goals for these patients (12).Conventional treatment for SHPT includes calcium supplementation, dietary phosphate restriction, oral phosphate-binding agents, active vitamin D sterols, and parathyroidectomy in patients refractory to therapy (13). Calcium supplement and calcium-containing phosphate binder use, however, induce hypercalcemia, particularly when administered concomitantly with vitamin D, which increases intestinal absorption of calcium and phosphorus. A significant proportion of hemodialysis patients receiving such treatment have elevated calcium and phosphorus, potentially requiring interruption of treatment that allows disease progression (8,13–15). Even in patients treated with paracalcitol that may have reduced capacity to enhance intestinal absorption of calcium and phosphorus, >60% experienced calcium levels >11 mg/dl and/or Ca × P >75 mg²/dl² (16–18). Consequently, achievement of KDOQI treatment goals is difficult with conventional treatment and novel treatment strategies are required.The calcimimetics have provided an alternative approach to the treatment of SHPT by directly targeting the calcium-sensing receptor (CaR) on the parathyroid cell surface that regulates the secretion of PTH. These agents increase the extracelluar calcium sensitivity of the CaR to lower circulating PTH levels within 1 to 2 h of administration (19–22). Cinacalcet (Sensipar/Mimpara, Amgen Inc., Thousand Oaks, CA) has been shown to significantly reduce PTH levels in dialysis patients with poorly controlled SHPT when added to conventional treatment regimens while simultaneously bringing about a reduction in serum calcium, phosphorus, and Ca × P (23–26).Administration of vitamin D sterols in most previously reported studies of cinacalcet was kept relatively constant to isolate the effect of cinacalcet (24–26). In the OPTIMA (Open-Label, Randomized Study Using Cinacalcet to Improve Achievement of KDOQI Targets in Patients with End-Stage Renal Disease) study, we compared a new treatment algorithm using cinacalcet and optimized doses of vitamin D, with the use of conventional therapy, to achieve KDOQI treatment goals for serum PTH, Ca × P, calcium, and phosphorus in patients with uncontrolled SHPT.     

Apparent mtDNA heteroplasmy in Alzheimer’s disease patients and in normals due to PCR amplification of nucleus-embedded mtDNA pseudogenes

In an unprecedented finding, Davis et al. [Davis, R. E., Miller, S., Herrnstadt, C., Ghosh, S. S., Fahy, E., Shinobu, L. A., Galasko, D., Thal, L. J., Beal, M. F., Howell, N. & Parker, W. D., Jr. (1997) Proc. Natl. Acad. Sci. USA 94, 4526–4531] used an unusual DNA isolation method to show that healthy adults harbor a specific population of mutated mitochondrial cytochrome c oxidase (COX) genes that coexist with normal mtDNAs. They reported that this heteroplasmic population was present at a level of 10–15% in the blood of normal individuals and at a significantly higher level (20–30%) in patients with sporadic Alzheimer’s disease. We provide compelling evidence that the DNA isolation method employed resulted in the coamplification of authentic mtDNA-encoded COX genes together with highly similar COX-like sequences embedded in nuclear DNA (“mtDNA pseudogenes”). We conclude that the observed heteroplasmy is an artifact.     

Stable transformation of the yellow fever mosquito, Aedes aegypti, with the Hermes element from the housefly

The mosquito Aedes aegypti is the world’s most important vector of yellow fever and dengue viruses. Work is currently in progress to control the transmission of these viruses by genetically altering the capacity of wild Ae. aegypti populations to support virus replication. The germ-line transformation system reported here constitutes a major advance toward the implementation of this control strategy. A modified Hermes transposon carrying a 4.7-kb fragment of genomic DNA that includes a wild-type allele of the Drosophila melanogaster cinnabar (cn) gene was used to transform a white-eyed recipient strain of Ae. aegypti. Microinjection of preblastoderm mosquito embryos with this construct resulted in 50% of the emergent G₀ adults showing some color in their eyes. Three transformed families were recovered, each resulting from an independent insertion event of the cn⁺-carrying transposon. The cn⁺ gene functioned as a semidominant transgene and segregated in Mendelian ratios. Hermes shows great promise as a vector for efficient, heritable, and stable transformation of this important mosquito vector species.