Tuning of CHO secretional machinery improve activity of secreted therapeutic sulfatase 150-fold.

Rare diseases are, despite their name, collectively common and millions of people are affected daily of conditions where treatment often is unavailable. Sulfatases are a large family of activating enzymes related to several of these diseases. Heritable genetic variations in sulfatases may lead to impaired activity and a reduced macromolecular breakdown within the lysosome, with several severe and lethal conditions as a consequence. While therapeutic options are scarce, treatment for some sulfatase deficiencies by recombinant enzyme replacement are available. The recombinant production of such sulfatases suffers greatly from both low product activity and yield, further limiting accessibility for patient groups. To mitigate the low product activity, we have investigated cellular properties through computational evaluation of cultures with varying media conditions and comparison of two CHO clones with different levels of one active sulfatase variant. Transcriptome analysis identified 18 genes in secretory pathways correlating with increased sulfatase production. Experimental validation by upregulation of a set of three key genes improved the specific enzymatic activity at varying degree up to 150-fold in another sulfatase variant, broadcasting general production benefits. We also identified a correlation between product mRNA levels and sulfatase activity that generated an increase in sulfatase activity when expressed with a weaker promoter. Furthermore, we suggest that our proposed workflow for resolving bottlenecks in cellular machineries, to be useful for improvements of cell factories for other biologics as well.

Screening of siblings to patients with abdominal aortic aneurysms in Sweden.

OBJECTIVE: The prevalence of Abdominal Aortic Aneurysm (AAA) is higher for First Degree Relatives to AAA-patients compared to the general population, regardless of sex. The prevalence of AAA is also higher in the North of Sweden compared to the Mid and South. A regional strong hereditary trait has been suggested as an explanation to this. The aim of this study was to investigate if siblings to AAA-patients in the North have a higher prevalence of AAA compared to siblings in the Mid-region. DESIGN: Cohort study. MATERIALS AND METHODS: All patients treated for AAA in a northern region (Norrbotten county, North) were screened for siblings. Consenting siblings, age 40-80, were examined (n = 379) with ultrasound. The results were compared to the previously published results of 150 ultrasound-screened siblings in the Mid-region (Stockholm county). RESULTS: The male/female ratio in the sibling cohort was 48% vs 52%. The prevalence of AAA in siblings in the North was 37/379 (brothers 14%, sisters 6%). This was not different from the prevalence among the Mid-region siblings 16/150 (brothers 17%, sisters 6% (p = 0.75). The distribution of risk factors was similar in the two regions. CONCLUSION: The results reinforce the importance of a more systematic approach towards selective screening of all siblings to AAA patients. Ultrasound should be performed in all eligible siblings, since the distribution of AAA is similar over regions. A correlation between the familial distribution and the reported high prevalence of AAA in general population in the North could not be shown.

Age at detection of abdominal aortic aneurysms in siblings of patients with abdominal aortic aneurysms.

OBJECTIVE: Few countries offer organized screening of siblings of patients with abdominal aortic aneurysms (AAAs), although a hereditary trait is well known to exist. Male relatives, but not female, are invited within the population-based screening programs for elderly men in Sweden. Evidence regarding the optimal age to screen siblings is scarce. The aim of this study was to describe the age at detection in siblings found with AAAs. METHODS: All patients treated for AAAs in two Swedish counties were screened for siblings. Consenting siblings aged 80 and younger were examined (N = 529) with ultrasound and were interviewed per protocol. RESULTS: In the cohort of 529 siblings to AAA patients, 53 siblings were diagnosed with AAAs (sisters 16/276 [5.8%] and brothers 37/253 [14.6%]). The prevalence of AAAs in the siblings 65 years of age or younger was 16/207 (7.7%). One-third of the siblings found with AAAs were young (16/53 [30%]). Among the young siblings with AAAs, 8/16 (50%) had an aneurysm larger than 50 mm or were already surgically treated. The prevalence of AAAs in siblings older than 65 years of age was 37/322 (12%). CONCLUSIONS: The AAA prevalence in this sibling cohort is strikingly high compared to the prevalence in the population (in Sweden, 1.4%-2.2% in 65-year-old men). The young ages among diagnosed siblings reinforce that male siblings of AAA patients should be screened before age 65 (before the population-based program) and that structured programs for female siblings are called for.

Effect of chronic mild stress on serotonergic markers in the skin and brain of the NC/Nga atopic-like mouse strain.

Atopic eczema is often worsened by stress. While acute stress is associated with increased turnover of serotonin (5-hydroxytryptamine; 5-HT), chronic stress causes a decrease. In chronic stress, there is a decrease of the 5-HT1A receptor (R)- and an increase in the 5-HT2AR-responsiveness to 5-HT. In the present study, the impact of chronic mild stress on the expression of 5-HT1A and 5-HT2A receptors and serotonin transporter protein (SERT) was investigated in eczematous skin and brain of atopic-like NC/Nga mice. Twenty-four NC/Nga mice were subjected to chronic mild stress for 12 weeks, and eczema was induced by applying a mite antigen (Dermatophagoides pteronyssinus) on the ears for the last 4 weeks. The mice were divided into three groups, eight per group, stressed eczematous (SE), non-stressed eczematous (NSE) and stressed control (SC). The biopsies were analysed by immunohistochemistry, using a streptavidin-biotin technique. There was an increased number of 5-HT containing dermal mast cell-like mononuclear cells in the skin of mice with eczema (SE and NSE, respectively) compared with the SC, and a tendency to more 5-HT-positive cells in the SE compared with the NSE group. Increased 5-HT1AR immunoreactivity (IR) in the skin and hippocampus of the eczematous groups compared to the control group was seen, but no difference between the SE and NSE groups. The epidermal immunoreactivity for 5-HT2AR was highest in the SE and NSE compared to the SC group, and was also higher in the SE compared to NSE. 5-HT2AR expression was also seen on nerve bundles, the number and intensity of such bundles being decreased in the SE compared to the NSE group. In the CA1 area of the hippocampus, there was an increase in the quantity of cells immunoreactive for 5-HT2AR in the SE versus the NSE group and also in the SE versus the SC group. SERT-IR was found also on nerve bundles with a decreased number in the SE compared to the NSE and SC group. There is a modulation of the expression of serotonergic markers in the eczematous skin and brain of the atopic-like mouse during chronic mild stress.

Local electronic structure of functional groups in glycine as anion, zwitterion, and cation in aqueous solution.

Nitrogen and oxygen K emission spectra of glycine in the form of anions, zwitterions, and cations in aqueous solution are presented. It is shown that protonation has a dramatic influence on the local electronic structure and that the functional groups give a distinct spectral fingerprint.

Electronic structure of water molecules confined in a micelle lattice.

Oxygen K absorption and emission spectra of water molecules confined in dodecyltrimethyl ammonium chloride micelle structures are presented. The local electronic structure of the water molecules is found to be dramatically different from the electronic structure of water molecules in the gas-phase as well as in liquid water. Hybridization with states of the ions in the surrounding ions is directly observed, and evidence for stabilization of the water molecules relative to molecules in bulk water is found.

Remote-controlled robotic arm for real-time echocardiography: the diagnostic future for patients in rural areas?

There exists a great clinical need for improving specialist consultation and utilization of echocardiography in areas remote from hospital-based care. This paper presents the development and first technical assessment of a concept of cardiovascular consultation utilizing long distance, real-time echocardiography as a diagnostic tool in rural areas. The development of CARdiological consultation at a DISTance (CARDISTA) was achieved in three stages, comprising tests of different broadband infrastructures, videoconference systems, microphones, cameras, monitors, and loudspeakers. The CARDISTA concept includes a cardiologist and a sonographer, a robotic arm (Medirob), a portable ultrasound machine, and presently available information technology using an advanced broadband backbone. The three stages provided, with some remaining doubts, echocardiographic examination at a distance comparable to hospital-based examinations. A continuous broadband capacity of 20 megabits per second (Mbps) seemed to be a vital component of CARDISTA for achieving the highest-quality imaging. With this broadband capacity, it was possible to achieve a transmission delay below 200 ms. The technical tests of the CARDISTA concept revealed promising results in enabling long distance real-time echocardiography for specialist consultation. CARDISTA is now ready for clinical testing and evaluation in rural areas for patients with heart diseases, especially heart failure.

Photoinduced formation of N2 molecules in ammonium compounds.

Via fluorescence yield (FY) and resonant inelastic scattering spectroscopy in the soft X-ray range we find that soft X-rays induce formation of N2 molecules in solid NH4Cl and in related compounds. The nitrogen molecules form weak bonds in NH4Cl, so that a substantial fraction of the molecules remains in the sample. From measurements of the FY as a function of exposure and temperature, the rates for the photochemical processes are estimated. At elevated temperatures (363 K), several nitrogen atoms are removed from the sample per incoming photon. At lower temperatures (233 K), the rate is reduced to around 0.02 nitrogen atoms for each incoming photon. Virtually all these atoms form N2 molecules which are bound in the sample. The generality and implications of these results are briefly discussed.