Reactivation of γ-globin in adult ß-YAC mice after ex vivo and in vivo hematopoietic stem cell genome editing.

Disorders involving ß-globin gene mutations, primarily ß-thalassemia and sickle cell disease, represent a major target for hematopoietic stem/progenitor cell (HSPC) gene therapy. This includes CRISPR/Cas9-mediated genome editing approaches in adult CD34+ cells aimed toward the reactivation of fetal γ-globin expression in red blood cells. Because models involving erythroid differentiation of CD34+ cells have limitations in assessing γ-globin reactivation, we focused on human ß-globin locus-transgenic (ß-YAC) mice. We used a helper-dependent human CD46-targeting adenovirus vector expressing CRISPR/Cas9 (HDAd-HBG-CRISPR) to disrupt a repressor binding region within the γ-globin promoter. We transduced HSPCs from ß-YAC/human CD46-transgenic mice ex vivo and subsequently transplanted them into irradiated recipients. Furthermore, we used an in vivo HSPC transduction approach that involves HSPC mobilization and the intravenous injection of HDAd-HBG-CRISPR into ß-YAC/CD46-transgenic mice. In both models, we demonstrated efficient target site disruption, resulting in a pronounced switch from human ß- to γ-globin expression in red blood cells of adult mice that was maintained after secondary transplantation of HSPCs. In long-term follow-up studies, we did not detect hematological abnormalities, indicating that HBG promoter editing does not negatively affect hematopoiesis. This is the first study that shows successful in vivo HSPC genome editing by CRISPR/Cas9.

Study of chloroquine susceptibility potential of plants using pseudomonas aeruginosa as in vitro model.

Chloroquine (CQ) is mainly known for antimalarial activity but due to lower sensitivity, it has not been well explored in the microbial disease treatment. In the present investigation, we attempted to enhance the CQ sensitivity in Pseudomonas aeruginosa. Presence of efflux pump is well demonstrated in bacterial system which plays an important role in drug sensitivity and resistance in bacteria and also serves other functions. Taking the advantage of presence of efflux pump in Pseudomonas aeruginosa, we made an attempt to sensitize the Pseudomonas aeruginosa with various plant extracts and phytochemicals for the development of CQ sensitivity. Ten rationally selected plant extracts were screened for the development of chloroquine sensitivity in P. aeruginosa. The chloroquine susceptibility assay was demonstrated by combining CQ and verapamil (a known efflux pump inhibitor) as a standard in an in vitro assay system. Results were quite encouraging as methanolic extracts of Syzygium aromaticum, Zingiber officinale and Curcuma longa were able to enhance chloroquine sensitivity in P. aeruginosa by increasing the zone of inhibition in well-defined assay system. These plant extracts were finally analysed for the presence of various phytochemicals. The Syzygium aromaticum extract showed the presence of phytochemicals, such as quinones, phenol, triterpenoid, saponins, tannins, alkaloids and flavonoids. On the other hand, the methanolic extract of Zingiber officinale and Curcuma longa showed the presence of saponins, tannins, alkaloids and flavonoids in the extract. Towards the identification of active principle of selected plant extract for CQ sensitivity enhancement, thin-layer chromatography was performed and various phytocomponent bands were isolated. Flavonoid (R f 0.44) in Syzygium aromaticum, alkaloid (R f 0.43) in Zingiber officinale and phenol (R f 0.62) in Curcuma longa were found responsible for the enhancement of CQ susceptibility in P. aeruginosa. This interesting finding confirmed the concept that a prior course or combination of plant extracts or phytochemicals with chloroquine can be effective against P. aeruginosa. Present investigation successfully presented the proof of concept for the enhancement of chloroquine sensitivity in bacterial system by modulating an efflux pump. Concept can be explored for repurposing chloroquine for new applications. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03382-1.

Evaluation of Early Knee Osteoarthritis Using Biomechanical and Biochemical Markers.

Altered cellular mechano-transduction and biochemistry lead to degeneration of articular cartilage in people with knee osteoarthritis. However, the influence of low-moderate exposure to weight-bearing activity such as squatting on cartilage metabolism has not been adequately studied. The current study explored associations between knee adduction moment (KAM) during walking, biochemical markers and daily squat exposure. 3D gait analysis was used to determine external loads acting on the knee as indicators of joint compressive forces whereas biomarkers-Urine type-II-collagen-telopeptide (uCTxII), antioxidant and phospholipase A2 (PLA2) activity reflected on articular cartilage status. Following ethical approval, 66 participants with varying daily squat exposure (non-squatters [n = 21, exposure = 0 min]; activity of daily living [ADL] squatters [n = 16, exposure = 34 min]; occupational squatters [n = 13, exposure = 102 min]) and people with grade 2-3 knee osteoarthritis (n = 16, exposure = 28 min) were evaluated using 3D gait and biomarker analysis. The PLA2 activity was lowest in ADL squatters while occupational squatters demonstrated highest activity (p < 0.05). KAM and urine biomarker were similar among the groups. Moderate-strong positive association was observed between sweat PLA2 activity and age (r = 0.819, p = 0.004), daily squat exposure and biomarker uCTxII (r = 0.604, p = 0.013), antioxidant activity and Right-KAM (r = -0.917, p = 0.001), and Left-KAM (r = -0.767, p = 0.016), in people with knee OA. Healthy people demonstrated weak positive associations between KAM, uCTxII, and BMI. Associations between non-invasive biomechanical and biochemical markers indicate their potential use to identify early knee osteoarthritis. Studies with larger sample size are necessary to support prescription of body weight joint loading activities such as squatting in moderation, to delay functional decline caused by knee OA.