CRISPR-Cas12a-integrated transgenes in genomic safe harbors retain high expression in human hematopoietic iPSC-derived lineages and primary cells.

Discovery of genomic safe harbor sites (SHSs) is fundamental for multiple transgene integrations, such as reporter genes, chimeric antigen receptors (CARs), and safety switches, which are required for safe cell products for regenerative cell therapies and immunotherapies. Here we identified and characterized potential SHS in human cells. Using the CRISPR-MAD7 system, we integrated transgenes at these sites in induced pluripotent stem cells (iPSCs), primary T and natural killer (NK) cells, and Jurkat cell line, and demonstrated efficient and stable expression at these loci. Subsequently, we validated the differentiation potential of engineered iPSC toward CD34+ hematopoietic stem and progenitor cells (HSPCs), lymphoid progenitor cells (LPCs), and NK cells and showed that transgene expression was perpetuated in these lineages. Finally, we demonstrated that engineered iPSC-derived NK cells retained expression of a non-virally integrated anti-CD19 CAR, suggesting that several of the investigated SHSs can be used to engineer cells for adoptive immunotherapies.

Highly Selective Lysine Acylation in Proteins Using a Lys-His Tag Sequence.

Chemical modification of proteins has numerous applications, but it has been challenging to achieve the required high degree of selectivity on lysine amino groups. Recently, we described the highly selective acylation of proteins with an N-terminal Gly-His6 segment. This tag promoted acylation of the N-terminal Nα -amine resulting in stable conjugates. Herein, we report the peptide sequences Hisn -Lys-Hism , which we term Lys-His tags. In combination with simple acylating agents, they facilitate the acylation of the designated Lys Nϵ -amine under mild conditions and with high selectivity over native Lys residues. We show that the Lys-His tags, which are 7 to 10 amino acids in length and still act as conventional His tags, can be inserted in proteins at the C-terminus or in loops, thus providing high flexibility regarding the site of modification. Finally, the selective and efficient acylation of the therapeutic antibody Rituximab, pure or mixed with other proteins, demonstrates the scope of the Lys-His tag acylation method.

Fasting of male mice - Effects of time point of initiation and duration on clinical chemistry parameters and animal welfare.

Fasting of mice is a common procedure, which can affect the outcome of the study as well as animal welfare. In this study, we assess the effects of fasting, fasting duration and fasting initiation time in relation to light schedule and present suggestions for optimization of fasting. Male C57BL/6NCrl mice were fasted for 0, 3, 6, 12, 18 and 24 hours initiated either in the light period (photophase) or the dark period (scotophase). Body weight, gastric content, body temperature, corticosterone and 19 routine clinical chemistry parameters were evaluated. Fasting caused significant changes in most of the measured parameters. Increasing duration of fasting resulted in increasing physiological changes. Fasting initiated in the scotophase caused more significant changes than fasting initiated in the photophase. To cause the least physiological changes in mice and increase animal welfare, mice should preferably be fasted in the photophase and for the shortest possible period allowed by the experimental purpose of fasting.

Kettlebell swing targets semitendinosus and supine leg curl targets biceps femoris: an EMG study with rehabilitation implications.

BACKGROUND: The medial hamstring muscle has the potential to prevent excessive dynamic valgus and external rotation of the knee joint during sports. Thus, specific training targeting the medial hamstring muscle seems important to avoid knee injuries. OBJECTIVE: The aim was to investigate the medial and lateral hamstring muscle activation balance during 14 selected therapeutic exercises. STUDY DESIGN: The study design involved single-occasion repeated measures in a randomised manner. Sixteen female elite handball and soccer players with a mean (SD) age of 23 (3) years and no previous history of knee injury participated in the present study. Electromyographic (EMG) activity of the lateral (biceps femoris - BF) and medial (semitendinosus - ST) hamstring muscle was measured during selected strengthening and balance/coordination exercises, and normalised to EMG during isometric maximal voluntary contraction (MVC). A two-way analysis of variance was performed using the mixed procedure to determine whether differences existed in normalised EMG between exercises and muscles. RESULTS: Kettlebell swing and Romanian deadlift targeted specifically ST over BF (Δ17-22%, p<0.05) at very high levels of normalised EMG (73-115% of MVC). In contrast, the supine leg curl and hip extension specifically targeted the BF over the ST (Δ 20-23%, p<0.05) at very high levels of normalised EMG (75-87% of MVC). CONCLUSION: Specific therapeutic exercises targeting the hamstrings can be divided into ST dominant or BF dominant hamstring exercises. Due to distinct functions of the medial and lateral hamstring muscles, this is an important knowledge in respect to prophylactic training and physical therapist practice.