A retroviral expression system based on tetracycline-regulated tricistronic transactivator/repressor vectors for functional analyses of antiproliferative and toxic genes

Establishment of stably transfected mammalian cells with conditional expression of antiproliferative or proapoptotic proteins is often hampered by varying expression within bulk-selected cells and high background in the absence of the inducing drug. To overcome such limitations, we designed a gene expression system that transcribes the tetracycline-dependent rtTA2-M2-activator, TRSID-silencer, and selection marker as a tricistronic mRNA from a single retroviral vector. More than 92% of bulk-selected cells expressed enhanced green fluorescent protein or luciferase over more than three orders of magnitude in an almost linear, dose-dependent manner. To functionally test this system, we studied how dose-dependent expression of p27(Kip1) affects proliferation and viability of SH-EP neuroblastoma cells. Low to moderate p27(Kip1) expression caused transient G(0)-G(1) accumulation without reduced viability, whereas high p27(Kip1) levels induced significant apoptosis after 72 hours. This proves that this expression system allows concentration-dependent analysis of gene function and implicates p27(Kip1) as a critical regulator of both proliferation and apoptosis in SH-EP neuroblastoma cells.     

Sensitivity of Thoracic Digital Tomosynthesis (DTS) for the Identification of Lung Nodules

Thoracic computed tomography (CT) is considered the gold standard for detection lung pathology, yet its efficacy as a screening tool in regards to cost and radiation dose continues to evolve. Chest radiography (CXR) remains a useful and ubiquitous tool for detection and characterization of pulmonary pathology, but reduced sensitivity and specificity compared to CT. This prospective, blinded study compares the sensitivity of digital tomosynthesis (DTS), to that of CT and CXR for the identification and characterization of lung nodules. Ninety-five outpatients received a posteroanterior (PA) and lateral CXR, DTS, and chest CT at one care episode. The CXR and DTS studies were independently interpreted by three thoracic radiologists. The CT studies were used as the gold standard and read by a fourth thoracic radiologist. Nodules were characterized by presence, location, size, and composition. The agreement between observers and the effective radiation dose for each modality was objectively calculated. One hundred forty-five nodules of greatest diameter larger than 4 mm and 215 nodules less than 4 mm were identified by CT. DTS identified significantly more >4 mm nodules than CXR (DTS 32 % vs. CXR 17 %). CXR and DTS showed no significant difference in the ability to identify the smaller nodules or central nodules within 3 cm of the hilum. DTS outperformed CXR in identifying pleural nodules and those nodules located greater than 3 cm from the hilum. Average radiation dose for CXR, DTS, and CT were 0.10, 0.21, and 6.8 mSv, respectively. Thoracic digital tomosynthesis requires significantly less radiation dose than CT and nearly doubles the sensitivity of that of CXR for the identification of lung nodules greater than 4 mm. However, sensitivity and specificity for detection and characterization of lung nodules remains substantially less than CT. The apparent benefits over CXR, low cost, rapid acquisition, and minimal radiation dose of thoracic DTS suggest that it may be a useful procedure. Work-up of a newly diagnosed nodule will likely require CT, given its superior cross-sectional characterization. Further investigation of DTS as a diagnostic, screening, and surveillance tool is warranted.     

"Occupational backache" – surface electromyography demonstrates the advantage of an ergonomic versus a standard microscope workstation

Musculoskeletal symptoms such as low back pain, neck pain, and tension headache are reported by up to 80% of professional personnel involved in daily microscope work. Yet, in striking contrast to the high prevalence of complaints, there is a general unawareness of this issue both in those suffering, and those ordering and designing microscopes. We intend to call attention to this underestimated work-related health hazard and to demonstrate a potential means of prevention. We obtained repeated surface electromyographic (EMG) recordings from the most strained neck, upper limb, and back muscles in 12 healthy volunteers while they were operating a near-to-ergonomic prototype workstation and a conventional microscope, respectively. Mean EMG activity was reduced in all recorded muscles when operating the ergonomic workstation compared to the standard microscope. This improvement became more distinct with sustained work, and was most pronounced in those muscles displaying the highest degree of activity while using the standard microscope. We demonstrate the usefulness of surface EMG recordings to show the advantage of an ergonomically tailored and individually adjustable microscope workstation over a standard microscope. The former allows the operator to maintain a more physiological posture, and may thus prevent the development of cumulative musculoskeletal disorders during prolonged microscope-related work. Electronic Publication     

A novel sandwich ELISA for alpha1 domain based detection of soluble HLA-G heavy chains

The detection of soluble human leukocyte antigen G (HLA-G) has been a technically demanding task for several years now and various enzyme linked immunosorbent assay (ELISA) formats have been designed. However, no ELISA test has been described so far which is able to detect all possible kinds of soluble HLA-G (sHLA-G) molecules that might occur in bio fluids. Here we describe a new ELISA approach able to recognize soluble alpha1 domain containing heavy chains of all HLA-G isoforms. The detection limit is shown to be at about 150 pg soluble recombinant HLA-G1 heavy chain per milliliters. Detectable HLA-G fragments are shown to occur in the supernatants of different HLA-G transfected cell lines and appear to be particularly abundant in supernatant of trophoblast derived choriocarcinoma cell lines. The novel ELISA employs the well characterized HLA-G mAbs 4H84 and MEM-G1 which ensure high HLA-G specificity. A negative control ELISA format, designed against non-existing analytes, has been established to reveal non-specific signal interference.     

A rapid screening for steroid 21‐hydroxylase mutations in patients with congenital adrenal hyperplasia

Steroid 21‐hydroxylase deficiency is the major cause of congenital adrenal hyperplasia (CAH). CAH due to 21‐hydroxylase deficiency is divided into three classes: salt‐wasting (classical), non‐classical and simple virilizing, reflecting different degrees of clinical severity. Using polymerase chain reaction (PCR) and allele‐specific oligonucleotide hybridisation (ASO), we screened the DNA of 62 Caucasian CAH families (heterozygous parents and children) for 14 different and frequently‐found CYP21‐mutations (HGMD). Of the 62 patients (21 males, 41 females), 26 females and 11 males had the classical or salt‐wasting form, 3 females and 1 male had the non‐classical form and 14 females and 7 males had simple virilizing CAH. More than 60% of the patients were compound‐heterozygous. We found the mutations on 110 alleles (out of 124 alleles). There were 30 CYP21 gene deletions/conversions, 3 substitutions (P30L) in exon 1, 30 splice mutations (c.93‐13A/C>G) in intron 2, 26 point mutations (I172N) in exon 4, one cluster of mutations (I236N, V237E, M239K) in exon 6, 8 mutations (V281L and 1760‐1761insT) in exon 7, and 8 nonsense (Q318X) and 4 missense (R356W) mutations in exon 8. Our study supports the case for using this rapid technique for CAH‐family screening as long as alleles from both affected patients and parents are screened in parallel. Hum Mutat 13:505, 1999. © 1999 Wiley‐Liss, Inc.