Molecular basis for differential Igk versus Igh V(D)J joining mechanisms.

In developing B cells, V(D)J recombination assembles exons encoding IgH and Igκ variable regions from hundreds of gene segments clustered across Igh and Igk loci. V, D and J gene segments are flanked by conserved recombination signal sequences (RSSs) that target RAG endonuclease1. RAG orchestrates Igh V(D)J recombination upon capturing a JH-RSS within the JH-RSS-based recombination centre1-3 (RC). JH-RSS orientation programmes RAG to scan upstream D- and VH-containing chromatin that is presented in a linear manner by cohesin-mediated loop extrusion4-7. During Igh scanning, RAG robustly utilizes only D-RSSs or VH-RSSs in convergent (deletional) orientation with JH-RSSs4-7. However, for Vκ-to-Jκ joining, RAG utilizes Vκ-RSSs from deletional- and inversional-oriented clusters8, inconsistent with linear scanning2. Here we characterize the Vκ-to-Jκ joining mechanism. Igk undergoes robust primary and secondary rearrangements9,10, which confounds scanning assays. We therefore engineered cells to undergo only primary Vκ-to-Jκ rearrangements and found that RAG scanning from the primary Jκ-RC terminates just 8 kb upstream within the CTCF-site-based Sis element11. Whereas Sis and the Jκ-RC barely interacted with the Vκ locus, the CTCF-site-based Cer element12 4 kb upstream of Sis interacted with various loop extrusion impediments across the locus. Similar to VH locus inversion7, DJH inversion abrogated VH-to-DJH joining; yet Vκ locus or Jκ inversion allowed robust Vκ-to-Jκ joining. Together, these experiments implicated loop extrusion in bringing Vκ segments near Cer for short-range diffusion-mediated capture by RC-based RAG. To identify key mechanistic elements for diffusional V(D)J recombination in Igk versus Igh, we assayed Vκ-to-JH and D-to-Jκ rearrangements in hybrid Igh-Igk loci generated by targeted chromosomal translocations, and pinpointed remarkably strong Vκ and Jκ RSSs. Indeed, RSS replacements in hybrid or normal Igk and Igh loci confirmed the ability of Igk-RSSs to promote robust diffusional joining compared with Igh-RSSs. We propose that Igk evolved strong RSSs to mediate diffusional Vκ-to-Jκ joining, whereas Igh evolved weaker RSSs requisite for modulating VH joining by RAG-scanning impediments.

Homeopathy for seasonal allergic rhinitis: rationale, design and methods of the three-armed randomized controlled HOMEOSAR trial.

BACKGROUND: Patients with seasonal allergic rhinitis (SAR) frequently use homeopathic therapy. Although there is some evidence that homeopathy may be effective in treating symptoms of SAR, there is a lack of high-quality clinical trials. Therefore, the aim of the homeopathy for SAR (HOMEOSAR) trial is to determine the efficacy of individualized or standardized homeopathic drug treatment compared to placebo regarding rhinitis-related quality of life in patients with SAR. METHODS: This randomized, placebo-controlled, double-blind, three-armed intervention study will be conducted at two university hospital outpatient clinics for complementary and integrative medicine in Berlin and in 12 office-based practices specializing in homeopathic treatment in Germany. A total of 270 patients with clinical symptoms of SAR and positive allergy test to birch and grass pollen will receive homeopathic anamnesis and subsequently be randomized into (a) standardized homeopathic drug treatment with Galphimia Glauca (potency D6), (b) individualized homeopathic drug treatment (D6), or (c) placebo. All three groups can receive on-demand rescue medication as needed. Treatment will consist of two consultations and daily intake of the study medication for 4 weeks during the pollen season. The primary outcome is the mean overall score of the Rhinitis Quality of Life Questionnaire (RQLQ) in weeks 3 and 4, analyzed using analysis of covariance (adjusted for baseline RQLQ overall score and study center). A closed testing procedure will be used to control the overall type I error comparing the 3 treatment groups. Secondary outcomes include the overall RQLQ and its seven domain scores, responder status (decrease in RQLQ overall score of at least 0.5 points compared to the baseline value), use of rescue medication, intensity of total and individual SAR symptoms based on visual analog scale, generic health-related quality of life, safety, utilization of health care resources and associated costs. In addition, a qualitative data analysis is planned. CONCLUSION: The results of our study will contribute to clarifying the possible therapeutic effects of homeopathic drug treatment for patients with SAR. TRIAL REGISTRATION: This study has been registered in the German Clinical Trial Registry with trial ID DRKS00018081 on June 09, 2020.

Strong and selective inhibitors of hepatitis B virus replication among novel N4-hydroxy- and 5-methyl-beta-L-deoxycytidine analogues.

Novel N(4)-hydroxy- and 5-methyl-modified beta-L-deoxycytidine analogues were synthesized and evaluated as anti-hepatitis B virus (HBV) agents. Their in vitro efficiencies were investigated in HepG2.2.15 cells stably transfected with HBV. beta-L-2',3'-Didehydro-2',3'-dideoxy-N(4)-hydroxycytidine (beta-L-Hyd4C) was most effective in reducing secreted HBV DNA (50% effective concentration [EC(50)], 0.03 microM), followed by beta-L-2',3'-dideoxy-3'-thia-N(4)-hydroxycytidine (EC(50), 0.51 microM), beta-L-2',3'-dideoxy-N(4)-hydroxycytidine (EC(50), 0.55 microM), and beta-L-5-methyl-2'-deoxycytidine (EC(50), 0.9 microM). The inhibition of the presumed target, the HBV DNA polymerase, by the triphosphates of some of the beta-L-cytidine derivatives was also assessed. In accordance with the cell culture data, beta-L-Hyd4C triphosphate was the most active inhibitor, with a 50% inhibitory concentration of 0.21 microM. The cytotoxicities of some of the 4-NHOH-modified beta-L-nucleosides were dramatically lower than those of the corresponding cytidine analogues with the unmodified 4-NH(2) group. The 50% cytotoxic concentrations for beta-L-Hyd4C in HepG2 and HL-60 cells were 2,500 microM and 3,500 microM, respectively. In summary, our results demonstrate that at least beta-L-Hyd4C can be recommended as a highly efficient and extremely selective inhibitor of HBV replication for further investigations.

Protein and RNA of human telomerase as targets for modified oligonucleotides.

Chimeric oligodeoxynucleotides containing phosphorothioate and N3'-->P5' phosphoramidate linkages were synthesized. These oligomers show a high inhibitory activity against human telomerase.

Pre-epithelial mucus layer in the colon of conventional and germ-free rats.

The pre-epithelial mucus layer (PML) and epithelial mucins were studied by mucin histochemistry in 10 microns-thick celloidinstabilized cryostat sections in the proximal and distal colon of conventional and germ-free rats aged 120 and 350 days. No continuous PML was found in the proximal colon. A continuous mucus blanket, of fairly homogenous thickness, was observed in the distal colon, where the PML-thickness was 40 +/- 24 microns at 120 days of age and 44 +/- 22 microns at 350 days of age in conventional rats, and 25 +/- 17 microns (120 days) and 22 +/- 10 microns (350 days) in germ-free rats. The stainability of the PML by periodic acid-Schiff and Alcian Blue at pH 2.5 and 1.0 was stronger in conventional rats than in germ-free rats, indicating higher concentrations of mucosubstances and of acid and sulphated mucins, respectively. The PML of the conventional rat distal colon showed a stratified structure of up to eight sublayers. In the distal colon of germ-free rats, the whole gut wall thickness was reduced 47% compared to the conventional rat (germ-free; 185 +/- 73 microns, conventional: 350 +/- 115 microns). No stratification of the PML was observed. The presence of intestinal microflora obviously had a strong influence on the thickness, compactness, mucin content, mucin composition and structure of the pre-epithelial mucus layer.

Scanning electron microscopy of deposits remaining in soft contact lens polishing marks after cleaning.

Deposits are a complication of contact lens wear that have been associated with ocular diseases, including keratitis and giant papillary conjunctivitis. The purpose of this morphologic study was to describe the deposits on soft contact lenses fabricated by different surface manufacturing methods and to evaluate the ease of removal of deposits by surfactant cleaning. We studied the anterior surfaces of 30 soft contact lenses (10 lathe cut [polished]; 10 spin cast [unpolished], and 10 cast molded [unpolished]) of the same polymer and water content. All lenses were worn for 8 hours by two asymptomatic persons who did not routinely wear contact lenses. Lenses from one eye were immersed in glutaraldehyde, while lenses from the other eye were cleaned before immersion in glutaraldehyde. All lenses were then dehydrated in graded alcohols, critical-point-dried, and observed by scanning electron microscopy. The amount of deposit on the lens surface was judged by an individual who did not know the identity of the lens. All lenses showed similar types of deposits. At the end of 8 hours wear, all types of lens surfaces were covered with deposits. Cleaning removed some but not all deposits. The lens surfaces manufactured in such a way as to leave polishing or lathe marks showed deposits "heaped-up" on the marks before cleaning and remaining in the mark after cleaning. Polishing marks from lens fabrication may represent an increased risk for conjunctival insult from deposits remaining in the marks after cleaning.

Inhibition of HIV-associated reverse transcriptase by sugar-modified derivatives of thymidine 5'-triphosphate in comparison to cellular DNA polymerases alpha and beta.

The sugar-modified dTTP analogues 2',3'-didehydro-2',3'-dideoxy-thymidine 5'-triphosphate (ddeTTP), 2',3'-dideoxythymidine 5'-triphosphate (ddTTP), 3'-fluorothymidine 5'-triphosphate (FdTTP), and 3'-azidothymidine 5'-triphosphate (N3dTTP) are demonstrated to be very effective and selective inhibitors of the HIV-associated reverse transcriptase (HIV-RT). This conclusion is based on a comparison of the ID50 values of the compounds for the HIV-RT (ranging from 0.03 microM for ddeTTP to 0.1 microM for ddTTP) and the cellular DNA polymerase alpha (greater than 200 microM). DNA polymerase beta is partially affected by N3dTTP (ID50 = 31 microM) and by the other analogues (ID50 = 1-2.2 microM). FdTTP has proved as effective as N3dTTP (ID50 = 0.05 microM) in suppressing the HIV-RT activity. Kinetic analysis revealed for both dTTP analogues a competitive type of inhibition and the same K1 values (about 0.05 microM).

A computer controlled automated dispensor for the eluting, calibrating and dispensing of 99Tcm-pertechnetate.

This paper describes an automated 99Tcm-Dispensor, a computer controlled device for the programmed, automated elution of 99Tcm-sodium pertechnetate, its calibration, metered delivery and dilution. The elution can be programmed up to 7 days in advance. The total radioactivity of the eluate is assayed by a semiconductor counter integrated in the shielding of the pertechnetate reservoir. The volume of eluate actually available for dispensing is measured by capacitance and is monitored continuously. The specific concentration is calculated from total activity and volume, and is corrected for decay prior to each delivery. All activity bearing sections are shielded by lead-antimony covers specified to attenuate the radiation from generators up to 2 Ci 99Mo/99Tcm. The labelling vials are inserted into the dispensing chamber within a shielding vessel manually. The sodium pertechnetate solution is dispensed prior to the optional additional delivery of normal saline. The Tc-Dispensor maintains sterility, guards against accidental radioactive contamination, and is suited to reduce the radiation burden of personnel. All operations are recorded by the computer enabling the print-out of protocols. We found the Tc-Dispensor safe, precise and accurate.

Controlling air traffic with touch sensitive screen.

Modern air traffic control (ATC) systems are increasingly using computer generated radar displays to present targets, target information and other general information to control personnel. On the control side the radar operator uses devices such as rolling ball, joystick, keyboard or lightpen. A laboratory demonstration of a new touch sensitive control device in a simulated ATC system is described in some detail together with results. Ergonomic advantages and design problems are also discussed.