Transgene-free genome editing in marine algae by bacterial conjugation - comparison with biolistic CRISPR/Cas9 transformation.

The CRISPR/Cas9 technology has opened the possibility for targeted genome editing in various organisms including diatom model organisms. One standard method for delivery of vectors to diatom cells is by biolistic particle bombardment. Recently delivery by conjugation was added to the tool-box. An important difference between these methods is that biolistic transformation results in transgene integration of vector DNA into the algae genome, whereas conjugative transformation allows the vector to be maintained as an episome in the recipient cells. In this study, we have used both transformation methods to deliver the CRISPR/Cas9 system to the marine diatom Phaeodactylum tricornutum aiming to induce mutations in a common target gene. This allowed us to compare the two CRISPR/Cas9 delivery systems with regard to mutation efficiency, and to assess potential problems connected to constitutive expression of Cas9. We found that the percentage of CRISPR-induced targeted biallelic mutations are similar for both methods, but an extended growth period might be needed to induce biallelic mutations when the CRISPR/Cas9 system is episomal. Independent of the CRISPR/Cas9 vector system, constitutive expression of Cas9 can cause re-editing of mutant lines with small indels. Complications associated with the biolistic transformation system like the permanent and random integration of foreign DNA into the host genome and unstable mutant lines caused by constitutive expression of Cas9 can be avoided using the episomal CRISPR/Cas9 system. The episomal vector can be eliminated from the diatom cells by removal of selection pressure, resulting in transient Cas9 expression and non-transgenic mutant lines. Depending on legislation, such lines might be considered as non-GMOs.

Patients' safety, privacy and effectiveness--a conflict of interests in health care information systems?

Information technology (IT) is finding its way into daily clinical work. IT is primarily seen as a tool for providing quality of service, cutting costs and promoting efficiency in every aspect of health care, but improvements for patients' safety are also a driving force. IT-solutions can be found both at an administrative and a clinical level, supporting everything from documentation, distribution and storing of patient data to workflows, monitoring and decision making. However, the increasing use of IT in health care raises questions. What is the impact on patients' rights and privacy? Does the law benefit IT-solutions in health care, or does it raise barriers for optimized use? Which interests does the law safeguard in the health care sector, and in the light of an increasing use of IT, do any of these identified interests collide? In conjunction with a governmental national project (InfoVU) during 2001-2004, the Swedish National Board of Health and Welfare (NBHW) had to address these issues and other legal aspects of IT use in health care. The agency's analysis was published in November 2005. The purpose of this article is to present some of the agency's conclusions on legal issues pertaining to the management and processing of patient data. It will show, from a Swedish legislative point of view, the need for a common information security strategy for health care information management as well as discussing other legislative issues in order to meet both the patients' and the health care provider's interests in an electronic environment.

Predominance of detrimental humoral immune responses to HIV-1 in AIDS patients with CD4 lymphocyte counts less than 400/mm3.

The humoral immune response to human immunodeficiency virus type 1 (HIV-1) was studied in 25 AIDS patients with CD4 lymphocyte counts of less than 400/mm3. Humoral immune responses against tissue culture adapted strains of HIV-1, and two limited-passage patient isolates were investigated. Total anti-HIV antibody levels were not significantly different between different individuals. Neutralizing titres against HIVLA1 and HIVSF2 were 10- to 100-fold higher than against clinical isolates. The complement-mediated, antibody-dependent enhancement of HIV-1 infection titre was high (mean 1:14,000). Antibody-complement mediated cytotoxicity of both HIVLA1 and HIVSF2 was ineffective using human complement as a complement source. The antibody-dependent, cell-mediated cytotoxicity (ADCC) activity varied against the four isolates with tissue culture-adapted strains being more susceptible than clinical isolates. Finally, an ADCC effector cell function, natural killer or NK activity, was measured for all 25 patients, and NK activity of patients was decreased by nearly 75% compared to uninfected individuals. In summary, beneficial humoral immune responses are low in HIV-1 infected individuals with CD4 counts of less than 400/mm3 if the in vitro assay system is constructed to best mimic the in vivo situation. These results suggest that the lack of functional antibody responses to HIV may play an important role in viral pathogenesis.