OliTag-seq enhances in cellulo detection of CRISPR-Cas9 off-targets.

The potential for off-target mutations is a critical concern for the therapeutic application of CRISPR-Cas9 gene editing. Current detection methodologies, such as GUIDE-seq, exhibit limitations in oligonucleotide integration efficiency and sensitivity, which could hinder their utility in clinical settings. To address these issues, we introduce OliTag-seq, an in-cellulo assay specifically engineered to enhance the detection of off-target events. OliTag-seq employs a stable oligonucleotide for precise break tagging and an innovative triple-priming amplification strategy, significantly improving the scope and accuracy of off-target site identification. This method surpasses traditional assays by providing comprehensive coverage across various sgRNAs and genomic targets. Our research particularly highlights the superior sensitivity of induced pluripotent stem cells (iPSCs) in detecting off-target mutations, advocating for using patient-derived iPSCs for refined off-target analysis in therapeutic gene editing. Furthermore, we provide evidence that prolonged Cas9 expression and transient HDAC inhibitor treatments enhance the assay's ability to uncover off-target events. OliTag-seq merges the high sensitivity typical of in vitro assays with the practical application of cellular contexts. This approach significantly improves the safety and efficacy profiles of CRISPR-Cas9 interventions in research and clinical environments, positioning it as an essential tool for the precise assessment and refinement of genome editing applications.

Enhancing cord blood stem cell-derived NK cell growth and differentiation through hyperosmosis.

BACKGROUND: Natural killer (NK) cells hold great promise in treating diverse hematopoietic and solid tumors. Despite their availability from peripheral blood and cord blood, stem cell-derived NK cells offer an 'off-the-shelf' solution. Hematopoietic stem and progenitor cells (HSPCs) derived from cord blood pose no risk to the newborn or mother and are virtually ideal sources for NK cell differentiation. METHODS: We developed a modified protocol to differentiate HSPCs to NK cells under serum-free conditions using defined factors. The HSPC-derived NK (HSC-NK) cells could be expanded in a K562 feeder cell-dependent manner. Furthermore, using lentivirus transduction, chimeric antigen receptor (CAR)-modified HSPCs could be differentiated into NK cells, leading to the establishment of CAR-NK cells. RESULTS: The efficiency of NK cell differentiation from HSPCs was increased through the simple modulation of osmotic pressure by the addition of sodium chloride or glucose. Furthermore, the hyperosmosis-primed HSC-NK cells exhibited enhanced proliferation capacity and maintained normal functional characteristics, including transcriptome and antitumor efficacy. The optimized protocol yielded approximately 1.8 million NK cells from a single CD34-positive cell within a 28-day cycle, which signifies more than a ten-fold increase in efficiency relative to the conventional methods. This optimized protocol was also suitable for generating CAR-NK cells with high yields compared to standard conditions. CONCLUSIONS: The results of this study establish high osmotic pressure as a simple yet powerful adjustment that significantly enhances the efficiency and functionality of HSC-NK cells, including CAR-NK cells. This optimized protocol could lead to cost-effective, high-yield NK cell therapies, potentially revolutionizing cancer immunotherapy strategies.

Synthesis and Evaluation of Novel Nitric Oxide-Donating Ligustrazine Derivatives as Potent Antiplatelet Aggregation Agents.

Antiplatelet aggregation agents have demonstrated clinical benefits in the treatment of ischemic stroke. In our study, a series of novel nitric oxide (NO)-donating ligustrazine derivatives were designed and synthesized as antiplatelet aggregation agents. They were evaluated for the inhibitory effect on 5'-diphosphate (ADP)-induced and arachidonic acid (AA)-induced platelet aggregation in vitro. The results showed that compound 15d displayed the best activity in both ADP-induced and AA-induced assays, and compound 14a also showed quite better activity than ligustrazine. The preliminary structure-activity relationships of these novel NO-donating ligustrazine derivatives were discussed. Moreover, these compounds were docked with the thromboxane A2 receptor to study the structure-activity relationships. These results suggested that the novel NO-donating ligustrazine derivatives 14a and 15d deserve further study as potent antiplatelet aggregation agents.

[Effects of hypothermia on the repolarization duration of ventricular myocytes in rats and its mechanism].

Objective: To observe the effects of hypothermia on the repolarization duration and the expression of Kir2.1 protein of ventricular myocytes in isolated rat heart and explore the role of Kir2.1 protein.Methods: Eighteen healthy adult male Sprague-Dawley rats were randomly divided into three groups (n=6 per group): Control group (C group), 35℃ group (H1 group), 32℃ group (H2 group). Langendorff isolated heart models were established. After 15 min 37℃ K-H fluid banlanced perfusion, C group continued to perfuse the K-H solution at 37℃ for 30 minutes, H1 group continued to perfuse the K-H solution at 35℃ for 30 minutes, H2 group continued to perfuse the K-H solution at 32℃ for 30 minutes. At 15 min of balanced perfusion (T1), and 30 min of continuous perfusion (T2), the heart rate,and the MAP in the three layers of the left ventricular anterior wall were recorded, the action potential duration at 50% repolarization (MAPD50), the action potential duration at 90% repolarization (MAPD90) and transmural dispersion of repolarization(TDR) were calculated. At the same time, the occurrence of arrhythmia was recorded. The expression of Kir2.1 protein was measured by Western blot. The average optical density (AOD) and the distribution of Kir2.1 protein were measured by immunohistochemistry in the ventricular tissue measured by electrophysiology. Results: Compared with T0, the heart rate was decreased, MAPD50 and MAPD90 were prolonged significantly (P＜0.05), and TDR was increased significantly (P＜0.05) in H1 group, H2 group at T1. Compared with C group, the HR was decreased, the MAPD90 was prolonged significantly (P＜0.05), TDR was increased significantly (P＜0.05),the expression and the AOD of Kir2.1 protein were decreased significantly (P＜0.05) in H1group, H2group at T1. Compared with H1 group, the heart rate of H2 group was decreased significantly (P＜0.05), MAPD50 and MAPD90 were prolonged significantly (P＜0.05), and TDR was increased significantly (P＜0.05) at T1. The distribution of Kir2.1 protein in group C was normal, while the distribution of Kir2.1 in H1 group and H2 group was disordered. Conclusion: Hypothermia prolonged the ventricular duration of repolarization and increased the dispersion of repolarization. The mechanism is related to the down-regulation the expression of Kir2.1 protein and the disorder of the distribution of Kir2.1 protein.

Polysaccharides from loquat (Eriobotrya japonica) leaves: Impacts of extraction methods on their physicochemical characteristics and biological activities.

Impacts of hot water extraction (HWE), pressurized water extraction (PWE), high-speed shearing homogenization extraction, microwave assisted extraction (MAE), ultrasound assisted extraction (UAE), ultrasound assisted enzymatic extraction, and ultrasound-microwave assisted extraction (UMAE) on physicochemical characteristics and bioactivities of polysaccharides from loquat (Eriobotrya japonica) leaves (LLPs) were investigated. Results showed that the degrees of esterification, contents of phenolics and uronic acids, constituent monosaccharides, apparent viscosities, and molecular weights of LLPs varied by different extraction methods. Bioactivities of LLPs were also significantly affected by different extraction methods. The high molecular weight and high degree of esterification of LLP-W and LLP-P extracted by HWE and PWE, respectively, might contribute to their strong binding capacities. The strong antioxidant activities and inhibitory effects on α-amylase and α-glucosidase were found in LLP-M and LLP-U extracted by MAE and UAE, respectively, which might be attributed to their contents of uronic acids, contents of total phenolics, and molecular weights. The low molecular weights and viscosities of LLP-U and LLP-UM extracted by UMAE might contribute to their strong prebiotic effects. These findings could provide scientific foundations for selecting appropriate extraction methods to obtain LLPs with desired bioactivities for applications in the pharmaceutical and functional food industries.

Comparison of structural characteristics and bioactivities of polysaccharides from loquat leaves prepared by different drying techniques.

In the present study, freeze drying, hot-air drying, vacuum drying, and microwave drying at the microwave powers of 400, 600, and 800 W, respectively, were utilized to dry loquat leaves for evaluating the effects of different drying techniques on the physicochemical structures and bioactivities of polysaccharides extracted from loquat leaves (LLPs). Results demonstrated that the physicochemical structures and bioactivities of LLPs significantly affected by different drying techniques. The degrees of esterification, molar ratios of constituent monosaccharides, contents of uronic acids, apparent viscosities, and molecular weights of LLPs were varied by different drying techniques. Additionally, LLPs, particularly LLP-M4 which extracted from loquat leaves prepared by microwave drying at the power of 400 W, exerted remarkable in vitro binding capacities, strong inhibitory effects on α-amylase and α-glucosidase, and obvious antioxidant activities. Results indicated that the microwave drying could be an efficient drying technique before extraction of bioactive LLPs, and LLPs had great potential applications in the functional food and pharmaceutical industries.

Structural characteristics, rheological properties, and biological activities of polysaccharides from different cultivars of okra (Abelmoschus esculentus) collected in China.

In order to well understand the physicochemical characteristics and biological activities of polysaccharides (OPPs) from different cultivars of okra collected in China, the chemical characteristics, rheological properties, antioxidant activities, in vitro binding properties, and in vitro inhibitory effects on α-glucosidase of polysaccharides from five representative okra cultivars, including 'Lvjian', 'Kalong8', 'Shuiguo', 'Taiwanwufu', and 'Kalong3', were investigated and compared. Results showed that the constituent monosaccharides of OPPs were similar, which composed of rhamnose, galacturonic acid, galactose, and arabinose. However, their weight-average molecular weights varied from 2.76 × 103 to 4.20 × 103 kDa, and from 0.11 × 103 to 0.90 × 103 kDa, respectively. The uronic acids and degrees of esterification of OPPs ranged from 39.32% to 61.68%, and from 21.66% to 30.02%, respectively. OPPs exhibited typical shear-thinning behavior and viscoelastic properties. Furthermore, OPPs exhibited remarkable antioxidant activities, in vitro binding capacities, and inhibitory effects on α-glucosidase, which might be attributed to their relatively high content of uronic acids, high degrees of esterification, and high molecular weights. Results are helpful for better understanding of the physicochemical structures and bioactivities of OPPs, and OPPs had good application prospects as functional food ingredients for industrial applications.

[Value of multi-detector row CT and magnetic resonance imaging in the diagnosis of retinal detachment].

OBJECTIVE: To summarize multi-detector row CT (MDCT) and magnetic resonance imaging (MRI) features of retinal detachment and evaluate the diagnostic value of these two imaging modalities. METHODS: The MDCT and MRI manifestations were reviewed in 45 cases (47 eyes) of retinal detachment, among which 16 cases (17 eyes) were examined by MDCT and 29 cases (30 eyes) by MRI. Thirty-two cases (33 eyes) were confirmed by operation, and the other 13 cases (14 eyes) were confirmed based on the clinical findings. RESULTS: MDCT and MRI displayed signs of fluid retention between the detached retina and the posterior wall of the eyeball in the cases. Among all these cases, 21 eyes showed simple retinal detachment and 26 had also other pathologies (hemorrhage in 20 eyes and calcification in 6 eyes). Choroidal osteoma was identified in 3 eyes and melanoma of choroid in 5 eyes. CONCLUSION: MDCT is sensitive in detecting calcification in the eyes and MRI with a minimal risk of radiation, and shows advantages in displaying hemorrhage and confined retinal detachment. Both MDCT and MR have high clinical value in the diagnosis of retinal detachment, and their choice depends on the individual condition of the patients.

Esthesioneuroblastoma methods of intracranial extension: CT and MR imaging findings.

INTRODUCTION: Esthesioneuroblastoma (ENB) is an aggressive neuroectodermal malignancy in the upper nasal cavity with local infiltration and lymphatic or hematogenous metastasis. The purpose of this paper is to document three types of direct intracranial extensions by ENB using computed tomography (CT) and magnetic resonance imaging (MRI). METHODS: Eleven patients with pathologically confirmed ENB were admitted in our hospital between December 2002 and December 2008. Their magnetic resonance (MR; n = 10) and CT (n = 8) images were retrospectively reviewed, and particular attention was paid to tumor location and extension, enhancement pattern, cervical lymph node metastasis, and Kadish stage. RESULTS: The majority of patients were male (8/11) with Kadish stage C tumor (10/11). Three types of direct intracranial extension by ENBs were put forward according to their MR and CT findings. The primary tumors were well-defined soft-tissue masses centered in the roof of the nasal cavity eroding into the paranasal sinuses (11/11), the contralateral nasal cavity (4/11), the cranial cavity (5/11), and the fossa orbitalis (3/11). The tumor parenchyma were hypointensity on T1-weighted images, heterogeneous hyperintensity on T2-weighted images, and isodensity or slight hyperdensity on CT images with scattered necroses (4/11) and marginal cysts(4/11). Their enhancements were significant and inhomogeneous. Cervical lymph nodes metastases were observed in four patients (4/11), but no pathologically proved distant metastasis was observed. CONCLUSION: Three types of direct intracranial extensions by ENB can be found on CT and MRI: cranio-orbital-nasal-communicating ENB, cranio-nasal-communicating ENB, and orbital-nasal-communicating ENB.

[Magnetic resonance imaging findings of capillary hemangioma in the brain].

OBJECTIVE: To investigate the magnetic resonance imaging (MRI) findings of capillary hemangioma in the brain to improve the diagnosis of capillary hemangioma. METHODS: The MRI findings were analyzed in 6 patients with pathologically confirmed capillary hemangioma in the brain to define the characteristic MRI features of capillary hemangioma. RESULTS: In the 6 patients, the capillary hemangiomas were located in the pons (n=1), bulbus medullae (n=1), bilateral cerebellar hemisphere (n=1), right temporal lobe (n=1) or left frontal lobe (n=1). Three patients had subacute hemorrhage, 2 had cystic degeneration and 1 had solid tumors, all shoeing heterogeneous MRI signals. Contrast-enhanced MR scans revealed marked heterogeneous enhancement with clear boundaries of the tumor parenchyma, where spots and thin strips without enhancement were seen in 5 cases; vascular network was seen in 1 case. Drainage vessels connected to the lesions were found in 3 cases. CONCLUSION: MRI has a high diagnostic value for intracranial capillary hemangioma.

Separation and characterization of clindamycin phosphate and related impurities in injection by liquid chromatography/electrospray ionization mass spectrometry.

A simple high-performance liquid chromatography/electrospray ionization tandem mass spectrometric (HPLC/ESI-MS/MS) method has been developed for the rapid identification of clindamycin phosphate and its degradation products or related impurities in clindamycin phosphate injection. Detection was performed by quadrupole time-of-flight mass spectrometry (Q-TOFMS) via an ESI source in positive mode. Clindamycin phosphate and its related substances lincomycin, 7-epilincomycin-2-phosphate, lincomycin-2-phosphate, clindamycin B, clindamycin B-2-phosphate, and clindamycin were identified simultaneously by HPLC/ESI-MS/MS results. Based on the MS/MS spectra of their quasi-molecular ions, the fragmentation pathways of clindamycin phosphate and its related substances were compared and proposed, which are specific and useful for the identification of the lincosamide antibiotics and related impurities. The method was rapid, sensitive and specific and can be used to identify clindamycin phosphate and its related impurities in clindamycin phosphate injection without control compounds.

Separation and identification of purine nucleosides in the urine of patients with malignant cancer by reverse phase liquid chromatography/electrospray tandem mass spectrometry.

Urinary-modified nucleosides have a potential role as cancer biomarkers for a number of malignant diseases. High performance liquid chromatography (HPLC) was combined with full-scan mass spectrometry, MS/MS analysis and accurate mass measurements in order to identify purine nucleosides purified from urine. Potential purine nucleosides were assessed by their evident UV absorbance in the HPLC chromatogram and then further examined by the mass spectrometric techniques. In this manner, numerous modified purine nucleosides were identified in the urine samples from cancer patients including xanthine, adenosine, N1-methyladenosine, 5'-deoxy-5'-methylthioadenosine, 2-methyladenosine, N6-threonylcarbamoyladenosine, inosine, N1-methylinosine, guanosine, N1-methylguanosine, N7-methylguanine, N2-methylguanosine, N2,N2-dimethyguanosine, N2,N2,N7-trimethylguanosine. Furthermore, a number of novel purine nucleosides were tentatively identified via critical interpretation of the combined mass spectrometric data including N3-methyladenosine, N7-methyladenine, 5'-dehydro-2'-deoxyinosine, N3-methylguanine, O6-methylguanosine, N1,N2,N7-trimethylguanosine, N1-methyl-N2-ethylguanosine and N7-methyl-N1-ethylguanosine.

Analysis of modified nucleosides in the urine of patients with malignant cancer by liquid chromatography/electrospray ionization mass spectrometry.

As modified nucleosides reflect altered tRNA turnover which seems to be impaired in the body of cancer patients, they have been evaluated as potential tumor markers. High-performance liquid chromatography/electrosprary ionization quadrupole time-of-flight mass spectrometry (HPLC/ESI-Q-TOFMS) was used to identify nucleosides purified from urine in positive ionization mode. Potential nucleosides were assessed by their evident UV absorbance in HPLC and then further examined by mass spectrometric techniques. In this manner, 21 nucleosides were detected in the urine of a patient with lymphoid cancer including three modified nucleosides 5'-dehydro-2-deoxyinosine, N1,N2,N7-trimethylguanosine and N1-methyl-N2-ethylguanosine, which had never been reported previously.

Translation of SOX10 3' untranslated region causes a complex severe neurocristopathy by generation of a deleterious functional domain.

Peripheral demyelinating neuropathy, central dysmyelinating leukodystrophy, Waardenburg syndrome and Hirschsprung disease (PCWH) is a complex neurocristopathy caused by SOX10 mutations. Most PCWH-associated SOX10 mutations result in premature termination codons (PTCs), for which the molecular mechanism has recently been delineated. However, the first mutation reported to cause PCWH was a disruption of the native stop codon that by conceptual translation extends the protein into the 3' untranslated region (3'-UTR) for an additional 82 residues. In this study, we sought to determine the currently unknown molecular pathology for the SOX10 extension mutation using in vitro functional assays. Despite the wild-type SOX10 coding sequence remaining intact, the extension mutation led to severely diminished transcription and DNA-binding activities. Nevertheless, it showed no dominant-negative interference with wild-type SOX10 in vitro. Within the 82-amino acid tail, an 11-amino acid region (termed the WR domain) was responsible primarily for the deleterious properties of the extension. The WR domain, presumably forming an alpha-helix structure, inhibited SOX10 transcription activities if inserted in the carboxyl-terminal half of the protein. The WR domain can also affect other transcription factors with a graded effect when fused to the carboxyl termini, suggesting that it probably elicits a toxic functional activity. Together, molecular pathology for the SOX10 extension mutation is distinct from that of more common PTC mutations. Failure to properly terminate SOX10 translation causes the generation of a deleterious functional domain that occurs because of translation of the normal 3'-UTR; the mutant fusion protein causes a severe neurological disease.

In vitro synergy of baicalein and gentamicin against vancomycin-resistant Enterococcus.

BACKGROUND AND PURPOSE: Little is known about the possible synergism of baicalein, a bioactive flavone of Scutellariae radix (a Chinese herb), when used in conjunction with other antimicrobial agents against vancomycin-resistant Enterococcus (VRE). This in vitro study examined the possible synergism of the combination of baicalein and gentamicin against VRE. METHODS: Minimal inhibitory concentrations (MICs) of baicalein as well as gentamicin were determined against 39 clinical isolates of VRE by the agar dilution method. Synergistic activities were determined using the checkerboard method based on the fractional inhibitory concentration indices and also the time-kill method. Further time-kill studies were conducted with these two agents against one randomly chosen clinical isolate, VRE-096. RESULTS: Minimal concentrations inhibiting 50% (MIC(50)) and 90% (MIC(90)) of isolates for baicalein and gentamicin were all >256 microg/mL. Synergism between baicalein and gentamicin was demonstrated against four clinical isolates of VRE (VRE-70, VRE-940, VRE-096 and VRE-721). When approximately 5 x 10(5) colony-forming units/mL of VRE-096 was incubated with both baicalein at a concentration of 32 microg/mL (1/8 x MIC) and gentamicin at a concentration of 128 microg/mL (1/2 x MIC), there was an inhibitory effect against VRE that persisted for 48 h. At 48 h, the combination of baicalein and gentamicin at these respective concentrations resulted in a reduction of growth by approximately 2 orders of magnitude compared to that for the starting inoculum and by 3 orders of magnitude compared to that for baicalein alone, the more active single agent. CONCLUSION: This study demonstrated that baicalein and gentamicin can act synergistically in inhibiting VRE in vitro.