A recombineering pipeline to clone large and complex genes in Chlamydomonas

The ability to clone genes has greatly advanced cell and molecular biology research, enabling researchers to generate fluorescent protein fusions for localization and confirm genetic causation by mutant complementation. Most gene cloning is polymerase chain reaction (PCR)�or DNA synthesis-dependent, which can become costly and technically challenging as genes increase in size, particularly if they contain complex regions. This has been a long-standing challenge for the Chlamydomonas reinhardtii research community, as this alga has a high percentage of genes containing complex sequence structures. Here we overcame these challenges by developing a recombineering pipeline for the rapid parallel cloning of genes from a Chlamydomonas bacterial artificial chromosome collection. To generate fluorescent protein fusions for localization, we applied the pipeline at both batch and high-throughput scales to 203 genes related to the Chlamydomonas CO₂ concentrating mechanism (CCM), with an overall cloning success rate of 77%. Cloning success was independent of gene size and complexity, with cloned genes as large as 23 kb. Localization of a subset of CCM targets confirmed previous mass spectrometry data, identified new pyrenoid components, and enabled complementation of mutants. We provide vectors and detailed protocols to facilitate easy adoption of this technology, which we envision will open up new possibilities in algal and plant research.

A high-throughput system was developed to clone large, complex genes at high frequency and perform mutant complementation and protein tagging with a range of fluorophores in Chlamydomonas reinhardtii.     

Increasing daily feeding occasions in restricted feeding strategies does not improve performance or well being of fattening pigs

Background  

The natural feeding behaviour of the pig is searching for feed by rooting activities throughout the day; self-feeding pigs randomly space their eating and drinking periods throughout the day consuming ten to twelve meals per day. Pigs in conventional fattening pig production are normally fed 2–3 times daily with the feed consumed within 15 minutes. The aim of this study was to determine if more frequent feedings could improve the performance of conventionally kept fattening pigs.     

A severe form of human combined immunodeficiency due to mutations in DNA ligase IV

DNA ligase IV (LigIV) deficiency was identified as the molecular basis for a severe form of combined immunodeficiency in two microcephalic siblings with cellular radiosensitivity. In one patient the diagnosis was made directly after birth, allowing analysis of the role of LigIV in the development of specific immune cells. Absolute numbers of B cells were reduced 100-fold and alphabeta T cells 10-fold, whereas gammadelta T cells were normal. Spectratyping of all three cell populations showed a diverse repertoire, but sequencing of IgH V(D)J junctions revealed shorter CDR3 regions due to more extensive nucleotide deletions among D and J elements and fewer N nucleotide insertions. Clonal restriction of IgG-expressing, but not IgM-expressing, B cells and the lack of primary and secondary lymph node follicles indicated impaired class switch recombination. Observations in the older sibling showed that this rudimentary immune system was able to mount specific responses to infection. However, partial Ab responses and extensive amplification of gammadelta T cells could not prevent a life-threatening course of viral and bacterial infections, the development of an EBV-induced lymphoma, and immune dysregulation reflected by severe autoimmune cytopenia. Impaired generation of immune diversity under conditions of limited LigIV activity can cause a human SCID variant with a characteristic immunological phenotype.     

Individual Differences in the Attentional Blink: The Temporal Profile of Blinkers and Non-Blinkers

Background

When two targets are presented in close temporal succession, the majority of people frequently fail to report the second target. This phenomenon, known as the ‘attentional blink’ (AB), has been a major topic in attention research for the past twenty years because it is informative about the rate at which stimuli can be encoded into consciously accessible representations. An aspect of the AB that has long been ignored, however, is individual differences.

Methodology/Principal Findings

Here we compare a group of blinkers (who show an AB) and non-blinkers (who show little or no AB), and investigate the boundary conditions of the non-blinkers'' remarkable ability. Second, we directly test the properties of temporal selection by analysing response errors, allowing us to uncover individual differences in suppression, delay, and diffusion of selective attention across time. Thirdly, we test the hypothesis that information concerning temporal order is compromised when an AB is somehow avoided. Surprisingly, compared to earlier studies, only a modest amount of suppression was found for blinkers. Non-blinkers showed no suppression, were more precise in selecting the second target, and made less order reversals than blinkers did. In contrast, non-blinkers made relatively more intrusions and showed a selection delay when the second target immediately followed the first target (at lag 1).

Conclusion/Significance

The findings shed new light on the mechanisms that may underlie individual differences in selective attention. The notable ability of non-blinkers to accurately perceive targets presented in close temporal succession might be due to a relatively faster and more precise target selection process compared to large blinkers.     

Complications of Antiretroviral Therapy Initiation in Hospitalised Patients with HIV-Associated Tuberculosis

Background

HIV-associated tuberculosis is a common coinfection in Sub-Saharan Africa, which causes high morbidity and mortality. A sub-set of HIV-associated tuberculosis patients require prolonged hospital admission, during which antiretroviral therapy initiation may be required. The aim of this study was to document the causes of clinical deterioration of hospitalised patients with HIV-associated tuberculosis starting antiretroviral therapy in order to inform healthcare practice in low- to middle-income countries.

Methods

Prospective, observational cohort study of adult inpatients with HIV-associated tuberculosis starting antiretroviral therapy in a dedicated tuberculosis hospital in Cape Town, South Africa. Causes of clinical deterioration and outcome were recorded in the first 12 weeks of antiretroviral therapy. Patients with rifampicin-resistant tuberculosis were excluded.

Results

Between May 2009 and November 2010, 112 patients (60% female), with a median age of 32 years were enrolled. At baseline the median CD4 count was 55 cells/mm³ (IQR 31–106) and HIV viral load 5.6 log copies/mL. All patients had significant comorbidity: 82% were bed-bound, 65% had disseminated tuberculosis and 27% had central nervous system tuberculosis. Seventy six patients (68%) developed 144 clinical events after starting antiretroviral therapy. TB-IRIS, hospital-acquired infections and significant drug toxicities occurred in 42%, 20.5% and 15% of patients respectively. A new opportunistic disease occurred in 15% of patients and a thromboembolic event in 8%. Mortality during the 12 week period was 10.6%.

Conclusions

High rates of TB-IRIS, hospital-acquired infections and drug toxicities complicate the course of patients with HIV-associated tuberculosis starting antiretroviral therapy in hospital. Despite the high morbidity, mortality was relatively low. Careful clinical management and adequate resources are needed in hospitalised HIV-TB patients in the 1^st three months following ART initiation.     

Application of liquid chromatography-mass spectrometry to the investigation of poisoning by Oenanthe crocata

Liquid chromatography-mass spectrometry (LC-MS) analysis of methanol extracts of Oenanthe crocata roots revealed that oenanthotoxin co-eluted with another major polyalkyne, 2,3-dihydro-oenanthotoxin, using the existing high performance liquid chromatography (HPLC) method (isocratic elution from C18 with aqueous methanol) for investigating Oenanthe poisoning. Positive ES or APCI gave [(M+H)-H(2)O](+) and its methanol adduct as major ion species for oenanthotoxin, whereas 2,3-dihydro-oenanthotoxin formed [M+H](+) and its methanol adduct. The two polyalkynes could be chromatographically resolved on C18 by gradient elution with aqueous acetonitrile. This provides superior analysis for oenanthotoxin using HPLC with photodiode array (PDA) detection alone, but for LC-MS/MS aqueous acetonitrile was less suitable due to poor ionisation and, with APCI, an increase in the relative abundance of a [M-1](+) species, which could confuse compound assignment. HPLC-PDA and LC-MS/MS methods using an aqueous acetonitrile or aqueous methanol mobile phase, respectively, were successful when applied to the analysis of the stomach contents of a pony suspected to have eaten O. crocata. Relevant product ion spectra, by ion trap MS/MS, accurate mass data and complete sets of (1)H and (13)C NMR spectral assignments are given for the two compounds.     

Zinc hyperaccumulation substitutes for defense failures beyond salicylate and jasmonate signaling pathways of Alternaria brassicicola attack in Noccaea caerulescens

The hypothesis of metal defense as a substitute for a defective biotic stress signaling system in metal hyperaccumulators was tested using the pathosystem Alternaria brassicicola–Noccaea caerulescens under low (2 µM), medium (12 µM) and high (102 µM) Zn supply. Regardless the Zn supply, N. caerulescens responded to fungal attack with the activation of both HMA4 coding for a Zn transporter, and biotic stress signaling pathways. Salicylate, jasmonate, abscisic acid and indoleacetic acid concentrations, as well as biotic stress marker genes (PDF1.2, CHIB, LOX2, PR1 and BGL2) were activated 24 h upon inoculation. Based on the activation of defense genes 24 h after the inoculation an incompatible fungal–plant interaction could be predicted. Nonetheless, in the longer term (7 days) no effective protection against A. brassicicola was achieved in plants exposed to low and medium Zn supply. After 1 week the biotic stress markers were even further increased in these plants, and this compatible interaction was apparently not caused by a failure in the signaling of the fungal attack, but due to the lack of specificity in the type of the activated defense mechanisms. Only plants receiving high Zn exhibited an incompatible fungal interaction. High Zn accumulation in these plants, possibly in cooperation with high glucosinolate concentrations, substituted for the ineffective defense system and the interaction turned into incompatible. In a threshold‐type response, these joint effects efficiently hampered fungal spread and, consequently decreased the biotic stress signaling.     

Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes

Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually neglected. The study of water dynamics in enzymes and the associated thermodynamical consequences is highly complex and has involved computer simulations, nuclear magnetic resonance (NMR) experiments, and calorimetry. Water tunnels that connect the active site with the surrounding solvent are key to solvent displacement and dynamics. The protocol herein allows for the engineering of these motifs for water transport, which affects specificity, activity and thermodynamics. By providing a biophysical framework founded on theory and experiments, the method presented herein can be used by researchers without previous expertise in computer modeling or biophysical chemistry. The method will advance our understanding of enzyme catalysis on the molecular level by measuring the enthalpic and entropic changes associated with catalysis by enzyme variants with obstructed water tunnels. The protocol can be used for the study of membrane-bound enzymes and other complex systems. This will enhance our understanding of the importance of solvent reorganization in catalysis as well as provide new catalytic strategies in protein design and engineering.     

Sex differences in relationships between habitat use and reproductive performance in Soay sheep (Ovis aries)

The role of habitat use in generating individual variation in fitness has rarely been examined empirically in natural populations of long‐lived mammals, particularly for both sexes simultaneously. This is the case despite the increase in studies attempting to understand evolutionary change in such populations. Using data from the St. Kilda Soay sheep population, we quantified the association between lifetime reproductive performance (lifetime breeding and reproductive success) and the proportion of the home range covered by a key grass species, H. lanatus, for 490 females and 304 males. Increased H. lanatus cover was associated only with increased female lifetime reproductive success, but increased lifetime breeding success for both sexes, arising through increased male longevity and increased female fecundity. This work suggests that improved understanding of the causes and consequences of fitness differences will likely require us to better account for habitat‐derived individual variation, and to do so for the sexes appropriately.     

Melanocortin-4 receptors expressed by cholinergic neurons regulate energy balance and glucose homeostasis

Melanocortin-4 receptor (MC4R) mutations cause dysregulation of energy balance and hyperinsulinemia. We have used mouse models to study the physiological roles of extrahypothalamic MC4Rs. Re-expression of MC4Rs in cholinergic neurons (ChAT-Cre, loxTB MC4R mice) modestly reduced body weight gain without altering food intake and was sufficient to normalize energy expenditure and attenuate hyperglycemia and hyperinsulinemia. In contrast, restoration of MC4R expression in brainstem neurons including those in the dorsal motor nucleus of the vagus (Phox2b-Cre, loxTB MC4R mice) was sufficient to attenuate hyperinsulinemia, while the hyperglycemia and energy balance were not normalized. Additionally, hepatic insulin action and insulin-mediated suppression of hepatic glucose production were improved in ChAT-Cre, loxTB MC4R mice. These findings suggest that MC4Rs expressed by cholinergic neurons regulate energy expenditure and hepatic glucose production. Our results also provide further evidence of the dissociation in pathways mediating the effects of melanocortins on energy balance and glucose homeostasis.