Gain and loss of fluid in metamorphosing larvae of Manduca sexta

Larvae of Manduca sexta, (Sphingidae) increase their weight approx. 50% just before pupation and then secrete this fluid during the formation of their burrows. Time-lapse cinematography indicates that the fluid is ejected into the walls of the final burrow. It may offer some mechanical support; it is not particularly repellent to ants or mice, and it contains only small amounts of the alkaloids ingested from its preferred food plants. Comparison to other species indicates that the gain and loss of water is associated with burrowing behaviour; the fluid appears to be used in providing hydraulic pressure for burrowing, in forming and cementing the pupal chamber, and in acting as a CO₂ trap underground. The secretion is a hypertonic, highly alkaline solution containing KHCO₃ and small amounts of Na⁺, Ca²⁺, Mg²⁺, PO₄^?3 and some proteins. Haemolymph levels of K⁺ decrease, and those of Ca²⁺ increase, during the secretory phase. When radioactive calcium is injected into mature larvae, it appears promptly in the secretion. If however, the injection is given more than 24 hr before the animal begins secreting, then the calcium is bound to haemolymph protein and does not appear in the secretion.     

A redox trap to augment the intein toolbox

The unregulated activity of inteins during expression and consequent side reactions during work‐up limits their widespread use in biotechnology and chemical biology. Therefore, we exploited a mechanism‐based approach to regulate intein autocatalysis for biotechnological application. The system, inspired by our previous structural studies, is based on reversible trapping of the intein's catalytic cysteine residue through a disulfide bond. Using standard mutagenesis, the disulfide trap can be implemented to impart redox control over different inteins and for a variety of applications both in vitro and in Escherichia coli. Thereby, we first enhanced the output for bioconjugation in intein‐mediated protein ligation, also referred to as expressed protein ligation, where precursor recovery and product yield were augmented fourfold to sixfold. Second, in bioseparation experiments, the redox trap boosted precursor recovery and product yield twofold. Finally, the disulfide‐trap intein technology stimulated development of a novel bacterial redox sensor. This sensor reliably identified hyperoxic E. coli harboring mutations that disrupt the reductive pathways for thioredoxin and glutathione, against a background of wild‐type cells. Biotechnol. Bioeng. 2013; 110: 1565–1573. © 2012 Wiley Periodicals, Inc.     

Successful use of a novel lux® i-Amylose-1 chiral column for enantioseparation of “legal highs” by HPLC

Bath salts, fumigations, cleaners and air fresheners, behind these terms substances are hidden, which count as “Legal Highs”. These fancy names are used to pretend Legal Highs as harmless compounds, to circumvent legal regulations for marketing as well as to increase the sales. Besides classic illicit drugs of synthetic origin such as amphetamines, cocaine and MDMA, the trade of these compounds, also known as new psychoactive substances (NPS), is not uncommon today. In many countries, NPS are still not subject to drug control. Among them, there are stimulants such as new amphetamine derivatives or cathinones, which possess a chiral centre. Little is known about the fact that the two possible enantiomers may differ in their pharmacological effect. The aim of this study was to test a novel HPLC column for the enantioseparation of a set of 112 NPS coming from different chemical groups and collected by internet purchases during the years 2010–2018. The CSP, namely Lux® 5 μm i-Amylose-1, LC Column 250 x 4.6 mm, was run in normal phase mode under isocratic conditions, UV detection was performed at 245 nm and 230 nm, injection volume was 10 μl and flow rate was 1 ml/min. With a mobile phase consisting of n-hexane/isopropanol/diethylamine (90:10:0.1), herein, 79 NPS were resolved into their enantiomers successfully, for 37 of them baseline resolution was achieved. After increase of lipophily of the mobile phase to 99:1:0.1, another 27 compounds were baseline separated. It was found that all separated NPS are traded as racemic compounds.     

Purification of Transferrin and Albumin from Mouse Ascites Fluid

Mouse ascites fluid, which is readily obtained when cell lines and hybridomas are maintained in host mice, is a convenient source of several plasma proteins. This paper describes procedures for the purification of albumin and transferrin from mouse ascites fluid. Mouse transferrin was prepared from a 50–75% ammonium sulfate fraction of mouse ascites fluid by CM- and DEAE-cellulose chromatography. Mouse albumin was obtained by the same purification route, but required an additional chromatography step on Cibacron Blue F3GA-agarose. Both proteins were shown to be homogeneous by polyacrylamide gel electrophoresis and Immunoelectrophoresis. Characterization, which included a determination of amino acid composition, partial N-terminal sequence, molecular weight and extinction coefficient, correlated well with known values reported for human transferrin and albumin. The purified mouse proteins may be useful for biochemical studies, antibody preparation, and as growth factors for hybridomas or other mouse cell lines maintained in culture.     

Reducing the Decline in Physical Activity during Pregnancy: A Systematic Review of Behaviour Change Interventions

Purpose

Physical activity (PA) typically declines throughout pregnancy. Low levels of PA are associated with excessive weight gain and subsequently increase risk of pre-eclampsia, gestational diabetes mellitus, hypertension disorders, delivery by caesarean section and stillbirth. Systematic reviews on PA during pregnancy have not explored the efficacy of behaviour change techniques or related theory in altering PA behaviour. This systematic review evaluated the content of PA interventions to reduce the decline of PA in pregnant women with a specific emphasis on the behaviour change techniques employed to elicit this change.

Search and Review Methodology

Literature searches were conducted in eight databases. Strict inclusion and exclusion criteria were employed. Two reviewers independently evaluated each intervention using the behaviour change techniques (BCT) taxonomy to identify the specific behaviour change techniques employed. Two reviewers independently assessed the risk of bias using the guidelines from the Cochrane Collaboration. Overall quality was determined using the GRADE approach.

Findings

A total of 1140 potentially eligible papers were identified from which 14 studies were selected for inclusion. Interventions included counselling (n = 6), structured exercise (n = 6) and education (n = 2). Common behaviour change techniques employed in these studies were goal setting and planning, feedback, repetition and substitution, shaping knowledge and comparison of behaviours. Regular face-to-face meetings were also commonly employed. PA change over time in intervention groups ranged from increases of 28% to decreases of 25%. In 8 out of 10 studies, which provided adequate data, participants in the intervention group were more physically active post intervention than controls.

Conclusions and Implications

Physical activity interventions incorporating behaviour change techniques help reduce the decline in PA throughout pregnancy. Range of behaviour change techniques can be implemented to reduce this decline including goals and planning, shaping knowledge and comparison of outcomes. A lack of high quality interventions hampers conclusions of intervention effectiveness.     

Severe Painful Vaso-Occlusive Crises and Mortality in a Contemporary Adult Sickle Cell Anemia Cohort Study

Background

Frequent painful vaso-occlusive crises (VOCs) were associated with mortality in the Cooperative Study of Sickle Cell Disease (CSSCD) over twenty years ago. Modern therapies for sickle cell anemia (SCA) like hydroxyurea are believed to have improved overall patient survival. The current study sought to determine the relevance of the association between more frequent VOCs and death and its relative impact upon overall mortality compared to other known risk factors in a contemporary adult SCA cohort.

Methods

Two hundred sixty four SCA adults were assigned into two groups based on patient reported outcomes for emergency department (ED) visits or hospitalizations for painful VOC treatment during the 12 months prior to evaluation.

Results

Higher baseline hematocrit (p = 0.0008), ferritin (p = 0.005), and HDL cholesterol (p = 0.01) were independently associated with 1 or more painful VOCs requiring an ED visit or hospitalization for acute pain. During a median follow-up of 5 years, mortality was higher in the ED visit/hospitalization group (relative risk [RR] 2.68, 95% CI 1.1-6.5, p = 0.03). Higher tricuspid regurgitatant jet velocity (TRV) (RR 2.41, 95% CI 1.5-3.9, p < 0.0001), elevated ferritin (RR 4.00, 95% CI 1.8-9.0, p = 0.001) and lower glomerular filtration rate (RR=2.73, 95% CI 1.6-4.6, p < 0.0001) were also independent risk factors for mortality.

Conclusions

Severe painful VOCs remain a marker for SCA disease severity and premature mortality in a modern cohort along with other known risk factors for death including high TRV, high ferritin and lower renal function. The number of patient reported pain crises requiring healthcare utilization is an easily obtained outcome that could help to identify high risk patients for disease modifying therapies.

Trial Registration

ClinicalTrials.gov NCT00011648 http://clinicaltrials.gov/     

Patchy deletion of Bmpr1a potentiates proximal pulmonary artery remodeling in mice exposed to chronic hypoxia

Reduced vascular expression of bone morphogenetic protein type IA receptor (Bmpr1a) has been found in patients with pulmonary arterial hypertension. Our previous studies in mice with patchy deletion of Bmpr1a in vascular smooth muscle cells and cardiac myocytes showed decreased distal vascular remodeling despite a similar severity of hypoxic pulmonary hypertension (HPH). We speculate increased stiffness from ectopic deposition of collagen in proximal pulmonary arteries might account for HPH. Pulsatile pressure-flow relationships were measured in isolated, ventilated, perfused lungs of SM22α;TRE-Cre;R26R;Bmpr1a ^flox/flox (KO) mice and wild-type littermates, following 21 days (hypoxia) and 0 days (control) of chronic hypoxia. Pulmonary vascular impedance, which yields insight into proximal and distal arterial remodeling, was calculated. Reduced Bmpr1a expression had no effect on input impedance Z ₀ (P = 0.52) or characteristic impedance Z _C (P = 0.18) under control conditions; it also had no effect on the decrease in Z ₀ via acute rho kinase inhibition. However, following chronic hypoxia, reduced Bmpr1a expression increased Z _C (P < 0.001) without affecting Z ₀ (P = 0.72). These results demonstrate that Bmpr1a deficiency does not significantly alter the hemodynamic function of the distal vasculature or its response to chronic hypoxia but larger, more proximal arteries are affected. In particular, reduced Bmpr1a expression likely decreased dilatation and increased stiffening in response to hypoxia, probably by collagen accumulation. Increased PA stiffness can have a significant impact on right ventricular function. This study illustrates for the first time how proximal pulmonary artery changes in the absence of distal pulmonary artery changes contribute to pulmonary arterial hypertension.     

Differential Regulation of Matrix-Metalloproteinases and Their Tissue Inhibitors in Patients with Aneurysmal Subarachnoid Hemorrhage

Background

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in vascular remodeling, (neuro)inflammation, blood-brain barrier breakdown and neuronal apoptosis. Proinflammatory mechanisms are suggested to play an important role during early brain injury and cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH). This study aimed to analyze MMP-3, MMP-9, TIMP-1 and TIMP-3 in patients with SAH and their respective association with cerebral vasospasm (CVS).

Methods

Blood samples were collected in 20 SAH patients on days 1 to 7, 9, 11, 13 and 15 and 20 healthy age and gender matched volunteers. Serum MMPs and TIMPs were analyzed using enzyme-linked immunosorbent assay. Doppler sonographic CVS was defined as a mean blood flow velocity above 120 cm/sec in the middle cerebral artery. When discharged from hospital and at 6 month follow-up neurological outcome was evaluated using the Glasgow Outcome Score and the modified Rankin Scale.

Results

MMP-9 was higher in SAH patients compared to healthy controls (p<0.001). Patients with CVS (n = 11) had elevated MMP-9 serum levels compared to patients without CVS (n = 9, p<0.05). Higher MMP-9 was observed in the presence of cerebral ischemia associated with cerebral vasospasm (p<0.05). TIMP-1 was increased in patients with SAH on day 4 (p<0.05). There was an imbalance of the MMP-9/TIMP-1 ratio in favor of MMP-9 in SAH patients, in particular those with CVS (p<0.001). MMP-3 and TIMP-3 were significantly lower in SAH patients throughout day 4 and day 7, respectively (p<0.05). We did not find an association between MMP-, TIMP levels and neurological outcome after 6 months.

Conclusions

MMP-3 and -9 are differentially regulated in SAH patients with both enzymes showing peak levels correlating with the development of CVS. The inhibitors TIMP-1 and -3 were low during the acute phase after SAH and increased later on which might suggest a preponderance of pro-inflammatory mechanisms.