Haplotyping cryptic Adélie penguin taxa using low-cost,high-resolution melt curves

Distinguishing morphologically cryptic taxa, by definition, requires genetic data such as DNA sequences. However, DNA sequences may not be obtained easily for taxa from remote sites. Here we provide the details of a high-resolution melt-curve-based method using taxon-specific primers that can distinguish two taxa of Adélie penguins, and that will be usable in Antarctica when combined with some of the newly developed field-deployable thermal cyclers. We suggest that the wider adoption of field-deployable polymerase-chain-reaction-based techniques will enable faster assignation of haplotype to individuals in situ, and so allow the targeting of observations and sample collection to specimens relevant to the research question. Targeting individuals will also reduce the need to repeatedly handle animals and reduce the time and travel required to complete field work.     

Exploring the linkage between cell culture process parameters and downstream processing utilizing a plackett‐burman design for a model monoclonal antibody

Linkage of upstream cell culture with downstream processing and purification is an aspect of Quality by Design crucial for efficient and consistent production of high quality biopharmaceutical proteins. In a previous Plackett‐Burman screening study of parallel bioreactor cultures we evaluated main effects of 11 process variables, such as agitation, sparge rate, feeding regimens, dissolved oxygen set point, inoculation density, supplement addition, temperature, and pH shifts. In this follow‐up study, we observed linkages between cell culture process parameters and downstream capture chromatography performance and subsequent antibody attributes. In depth analysis of the capture chromatography purification of harvested cell culture fluid yielded significant effects of upstream process parameters on host cell protein abundance and behavior. A variety of methods were used to characterize the antibody both after purification and buffer formulation. This analysis provided insight in to the significant impacts of upstream process parameters on aggregate formation, impurities, and protein structure. This report highlights the utility of linkage studies in identifying how changes in upstream parameters can impact downstream critical quality attributes. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:163–170, 2017     

Accuracy of diagnostic registers and management of chronic obstructive pulmonary disease: the Devon primary care audit

Background

Guidelines on COPD diagnosis and management encourage primary care physicians to detect the disease at an early stage and to treat patients according to their condition and needs. Problems in guideline implementation include difficulties in diagnosis, using spirometry and the disputed role of reversibility testing. These lead to inaccurate diagnostic registers and inadequacy of administered treatments. This study represents an audit of COPD diagnosis and management in primary care practices in Devon.

Methods

Six hundred and thirty two patients on COPD registers in primary care practices were seen by a visiting Respiratory Specialist Nurse. Diagnoses were made according to the NICE guidelines. Reversibility testing was carried out either routinely or based on clinical indication in two sub-samples. Dyspnoea was assessed. Data were entered into a novel IT-based software which computed guideline-based treatment recommendations. Current and recommended treatments were compared.

Results

Five hundred and eighty patients had spirometry. Diagnoses of COPD were confirmed in 422 patients (73%). Thirty nine patients were identified as asthma only, 94 had normal spirometry, 23 were restrictive and 2 had a cardiac disorder. Reversibility testing changed diagnosis of 11% of patients with airflow obstruction, and severity grading in 18%. Three quarters of patients with COPD had been offered practical help with smoking cessation. Short and long-acting anticholinergics and long acting beta-2 agonists had been under-prescribed; in 15–18% of patients they were indicated but not received. Inhaled steroids had been over-prescribed (recommended in 17%; taken by 60%), whereas only 4% of patients with a chronic productive cough were receiving mucolytics. Pulmonary rehabilitation was not available in some areas and was under-used in other areas.

Conclusion

Diagnostic registers of COPD in primary care contain mistakes leading to inaccurate prevalence estimates and inappropriate treatment decisions. Use of pre-bronchodilator readings for diagnosis overestimates the prevalence and severity in a significant minority, thus post bronchodilator readings should be used. Management of stable COPD does often not correspond to guidelines. The IT system used in this study has the potential to improve diagnosis and management of COPD in primary care.     

Phylogenetic studies of the genus <Emphasis Type="Italic">Cebus</Emphasis>(Cebidae-Primates) using chromosome painting and G-banding

Background  

Chromosomal painting, using whole chromosome probes from humans and Saguinus oedipus, was used to establish karyotypic divergence among species of the genus Cebus, including C. olivaceus, C. albifrons, C. apella robustus and C. apella paraguayanus. Cytogenetic studies suggested that the species of this genus have conservative karyotypes, with diploid numbers ranging from 2n = 52 to 2n = 54.     

Longitudinal study of Plasmodium falciparum and Plasmodium vivax in a Karen population in Thailand

Background

Pregnancy malaria is caused by Plasmodium falciparum -infected erythrocytes binding the placental receptor chondroitin sulfate A (CSA). This results in accumulation of parasites in the placenta with severe clinical consequences for the mother and her unborn child. Women become resistant to placental malaria as antibodies are acquired which specifically target the surface of infected erythrocytes binding in the placenta. VAR2CSA is most likely the parasite-encoded protein which mediates binding to the placental receptor CSA. Several domains have been shown to bind CSA in vitro; and it is apparent that a VAR2CSA-based vaccine cannot accommodate all the CSA binding domains and serovariants. It is thus of high priority to define minimal ligand binding regions throughout the VAR2CSA molecule.

Methods

To define minimal CSA-binding regions/peptides of VAR2CSA, a phage display library based on the entire var2csa coding region was constructed. This library was screened on immobilized CSA and cells expressing CSA resulting in a limited number of CSA-binding phages. Antibodies against these peptides were affinity purified and tested for reactivity against CSA-binding infected erythrocytes.

Results

The most frequently identified phages expressed peptides residing in the parts of VAR2CSA previously defined as CSA binding. In addition, most of the binding regions mapped to surface-exposed parts of VAR2CSA. The binding of a DBL2X peptide to CSA was confirmed with a synthetic peptide. Antibodies against a CSA-binding DBL2X peptide reacted with the surface of infected erythrocytes indicating that this epitope is accessible for antibodies on native VAR2CSA on infected erythrocytes.

Conclusion

Short continuous regions of VAR2CSA with affinity for multiple types of CSA were defined. A number of these regions localize to CSA-binding domains and to surface-exposed regions within these domains and a synthetic peptide corresponding to a peptide sequence in DBL2 was shown to bind to CSA and not to CSC. It is likely that some of these epitopes are involved in native parasite CSA adhesion. However, antibodies directed against single epitopes did not inhibit parasite adhesion. This study supports phage display as a technique to identify CSA-binding regions of large proteins such as VAR2CSA.     

Lack of Adaptation of Chimeric GB Virus B/Hepatitis C Virus in the Marmoset Model: Possible Effects of Bottleneck

Approximately 3% of the world population is chronically infected with hepatitis C virus (HCV). GB virus B (GBV-B), a surrogate model for HCV, causes hepatitis in tamarins and is the virus phylogenetically most closely related to HCV. Previously we described a chimeric GBV-B containing an HCV insert from the 5′ noncoding region (NCR) that was adapted for efficient replication in tamarins (Saguinus species). We have also demonstrated that wild-type (WT) GBV-B rapidly adapts for efficient replication in a closely related species, the common marmoset (Callithrix jacchus). Here, we demonstrate that the chimeric virus failed to adapt during serial passage in marmosets. The chimeric virus was passaged four times through 24 marmosets. During passage, two marmoset phenotypes were observed: susceptible and partially resistant. Although appearing to adapt in a resistant animal during a prolonged and gradual increase in viremia, the chimeric GBV-B failed to replicate efficiently upon passage to a naïve marmoset. The resistance was specific to the chimeric virus, as the chimeric virus-resistant animals were susceptible to marmoset-adapted WT virus during rechallenge studies. Three isolates of the chimeric virus were sequenced, and 20 nucleotide changes were observed, including eight amino acid changes. Three unique changes were observed in the 5′ NCR chimeric insert, an area that is highly conserved in HCV. We speculate that the failure of the chimeric virus to adapt in marmosets might be due to a bottleneck that occurs at the time of infection of resistant animals, which may lead to a loss of fitness upon serial passage.Worldwide, approximately 170 million people are chronically infected with hepatitis C virus (HCV). The current approved therapy involves the combination of pegylated alpha interferon and ribavirin and has response rates for sustained viral clearance of 42% and 82% for genotypes 1 and 2/3, respectively (15, 29). However, a significant proportion of the population still develops serious disease as a consequence of HCV infection. HCV infection is the leading cause for liver transplantation in the United States (1, 50), and liver cancer due to HCV infection is one of the most rapidly increasing types of cancer in the United States (20).GB virus B (GBV-B) is a hepatotropic virus that causes hepatitis in tamarins and is the virus phylogenetically most closely related to HCV (33, 36, 44), and as such, GBV-B represents an important small-primate surrogate model for HCV infections. The history of the GB agent is complex and originates with the inoculation of tamarins with serum obtained from a surgeon with hepatitis (for a review, see reference 3); however there is little doubt that GBV-B is a tamarin virus, despite the fact that it has never been isolated from tamarins a second time. GBV-B has a very narrow host range for tamarins, marmosets, and other closely related New World monkeys (6, 23, 54). The GBV-B model overcomes a number of limitations encountered when working with HCV (3, 22). Due to the limited availability of tamarins, our lab and others (5, 16, 21, 23) initiated GBV-B studies in common marmosets (Callithrix jacchus), a small New World primate closely related to the tamarin (Saguinus sp.). The marmoset and tamarin represent less expensive, more readily available, and smaller animal models than the chimpanzee. While replication in marmosets is typically higher than what is observed in HCV-infected chimpanzees, reproducible infection profiles require some adaptation to this host (5, 54). Although robust replication of HCV in vitro is now possible using specific adapted strains of HCV (derivatives of the JFH1 and H77-S) and the Huh-7.5 cell line (4, 27, 52, 55, 56), the GBV-B primary hepatocyte culture system (2) may be more suitable for some studies, especially those involving specific aspects of the innate immune response and other viral host interactions.The organization of the GBV-B genome is very similar to that of HCV and the GBV-B polyprotein gene encodes 10 proteins analogous to the HCV proteins. The polyprotein of GBV-B has approximately 25 to 30% homology to that of HCV at the amino acid level (33), while the 5′ and 3′ noncoding regions (NCRs) are more divergent (7, 33, 40). The HCV and GBV-B 5′ NCRs are essential for both replication and translation. The structures are similar; however, GBV-B domain I is predicted to fold into two stem-loops (SL), compared to one SL in HCV, and the GBV-B 5′ NCR is longer due mainly to additional SL IIB and IIC that are not present in HCV (Fig. ​(Fig.1A)1A) (40). The 5′ NCR contains the internal ribosomal entry site (IRES), which can directly bind the 40S ribosomal subunit in order to initiate translation of the viral RNA (19, 38). cis RNA elements involved in RNA replication are also located in the HCV 5′ NCR (for a review, see reference 49). In GBV-B, 5′ NCR segments essential for genome replication have recently been identified (53).Open in a separate windowFIG. 1.(A) Predicted structures of the GBV-B (left) and HCV (right) 5′ NCRs. The scissors on GBV-B represent the sequence that was excised and replaced by the HCV insert, which is represented by scissors on the HCV 5′ NCR. The locations of mutations detected during sequencing are boxed: the first box identifies the deletion (ΔC) in a run of cytosines, and the second box identifies the polymorphisms present at two adjacent uracils (C, C/U). (Adapted from reference 40.) (B) Schematic of the GBV-B genome. During passage of GB/III^HC in tamarins, nine mutations were identified in virus from the T2 serum used to inoculate M1, and these nine mutations remained fixed in all marmoset isolates sequenced. Amino acid changes are indicated by dark arrows, silent mutations are indicated by asterisks, and NCR changes are indicated by dotted arrows.The utility of the GBV-B model was increased by the development of infectious cDNA clones that induced hepatitis upon intrahepatic inoculation of tamarins with in vitro-transcribed RNA (7, 31, 45). In order to further increase the use of GBV-B as a model for HCV, chimeras between GBV-B and HCV were constructed (16, 43, 48). In one chimera, a portion of the GBV-B 5′ NCR containing domain III, which is within the IRES functional domain, was replaced by an analogous region of HCV (40-43). The chimeric GB/III^HC retained IRES translational function and supported replication in tamarins (43). In this study, we examined the host range of this chimeric virus during serial passage in marmosets. We found that chimeric GBV-B failed to adapt during passage in marmosets. Marmosets infected with GB/III^HC displayed variable phenotypes ranging from susceptible to resistant, which appear to be due to a polymorphism in the marmoset population that also affects wild-type (WT) GBV-B (54). The failure of chimeric virus to adapt to replication in marmosets with the resistant phenotype was specific to the chimeric virus, and not the WT, and may involve several factors, including reduced replication capacity and the requirement to acquire multiple adaptive mutations. These barriers cumulatively may result in GB/III^HC experiencing a bottleneck in the resistant marmoset host.     

Insulin-stimulated Phosphorylation of the Rab GTPase-activating Protein TBC1D1 Regulates GLUT4 Translocation

Insulin stimulates the translocation of the glucose transporter GLUT4 from intracellular locations to the plasma membrane in adipose and muscle cells. Prior studies have shown that Akt phosphorylation of the Rab GTPase-activating protein, AS160 (160-kDa Akt substrate; also known as TBC1D4), triggers GLUT4 translocation, most likely by suppressing its Rab GTPase-activating protein activity. However, the regulation of a very similar protein, TBC1D1 (TBC domain family, member 1), which is mainly found in muscle, in insulin-stimulated GLUT4 translocation has been unclear. In the present study, we have identified likely Akt sites of insulin-stimulated phosphorylation of TBC1D1 in C2C12 myotubes. We show that a mutant of TBC1D1, in which several Akt sites have been converted to alanine, is considerably more inhibitory to insulin-stimulated GLUT4 translocation than wild-type TBC1D1. This result thus indicates that similar to AS160, Akt phosphorylation of TBC1D1 enables GLUT4 translocation. We also show that in addition to Akt activation, activation of the AMP-dependent protein kinase partially relieves the inhibition of GLUT4 translocation by TBC1D1. Finally, we show that the R125W variant of TBC1D1, which has been genetically associated with obesity, is equally inhibitory to insulin-stimulated GLUT4 translocation, as is wild-type TBC1D1, and that healthy and type 2 diabetic individuals express approximately the same level of TBC1D1 in biopsies of vastus lateralis muscle. In conclusion, phosphorylation of TBC1D1 is required for GLUT4 translocation. Thus, the regulation of TBC1D1 resembles that of its paralog, AS160.Insulin stimulates glucose transport into adipose and muscle cells by increasing the amount of the GLUT4 glucose transporter at the cell surface by a process termed GLUT4 translocation (1, 2). Unstimulated adipocytes and myotubes sequester GLUT4 in intracellular compartments. Insulin activates signaling cascades that lead to the trafficking of specialized GLUT4 vesicles to the cell membrane and fusion of the vesicles therewith. A key signaling pathway for GLUT4 translocation proceeds from the insulin receptor through the activation of the protein kinase Akt. One Akt substrate that connects signaling to GLUT4 trafficking is the Rab GTPase-activating protein (GAP)³ known as AS160. There is now considerable evidence for the following scheme (2, 3): under basal conditions, AS160 acts as a brake on GLUT4 translocation by maintaining one or more Rab proteins required for translocation in their inactive GDP state; in response to insulin, Akt phosphorylates AS160 and thereby suppresses its GAP activity; as a consequence, the elevation of the GTP form of the Rab proteins occurs, leading to the increased docking and subsequent fusion of the GLUT4 vesicles at the plasma membrane.More recently, we and others have characterized a paralog of AS160 known as TBC1D1 (4–7). Overall, TBC1D1 is 47% identical to AS160, with the GAP domain being 79% identical (4). Its GAP domain has the same Rab specificity as the GAP domain of AS160 (4). TBC1D1 is predominantly expressed in skeletal muscle; its expression in adipocytes is very low (5, 6). Nevertheless, 3T3-L1 adipocytes are a convenient cell type in which to examine the role of proteins in GLUT4 translocation, because insulin causes an ∼10-fold increase in GLUT4 at the cell surface. Previously, we examined the role of TBC1D1 in GLUT4 translocation by overexpressing it in 3T3-L1 adipocytes. Surprisingly, even though insulin led to phosphorylation of TBC1D1 on Akt site(s), ectopic TBC1D1 potently inhibited GLUT4 translocation (4, 5). By contrast, overexpression of AS160 did not inhibit GLUT4 translocation (8). This difference suggested that the regulation of TBC1D1 might be fundamentally different from that of AS160. In the present study, we show that this is not the case. By reducing the level of ectopic TBC1D1, we have obtained evidence that phosphorylation of TBC1D1 on several likely Akt sites relieves the inhibitory effect on GLUT4 translocation. In addition, we have examined the effect of a variant of TBC1D1 genetically associated with obesity on GLUT4 translocation and determined the relative levels of TBC1D1 in muscle biopsies from healthy and type 2 diabetic individuals.