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.

Mean arterial pressure to norepinephrine equivalent dose ratio for predicting renal replacement therapy requirement: a retrospective analysis from the MIMIC-IV.

BACKGROUND: This study aimed to assess the predictive value of the ratio of mean arterial pressure (MAP) to the corresponding peak rate of norepinephrine equivalent dose (NEQ) within the first day in patients with shock for the subsequent renal replacement therapy (RRT) requirement. METHODS: Patients were identified using the Medical Information Mart for Intensive Care IV (MIMIC-IV) database. The relationship was investigated using a restricted cubic spline curve, and propensity score matching(PSM) was used to eliminate differences between groups. Odds ratios (OR) with 95% confidence intervals (CI) were calculated using logistic regression. Variable significance was assessed using extreme gradient boosting (XGBoost), and receiver operating characteristic (ROC) curves were generated. RESULTS: Of the 5775 patients, 301 (5.2%) received RRT. The MAP/NEQ index showed a declining L-shaped relationship for RRT. After PSM, the adjusted OR per 100 mmHg/mcg/kg/min for RRT was 0.93(95% CI 0.88-0.98). The most influential factors for RRT were fluid balance, baseline creatinine, and the MAP/NEQ index. The threshold for the MAP/NEQ index predicting RRT was 161.7 mmHg/mcg/kg/min (specificity: 65.8%, sensitivity: 74.8%) with an area under the ROC curve of 75.9% (95% CI 73.1-78.8). CONCLUSIONS: The MAP/NEQ index served as an alternative predictor of RRT necessity based on the NEQ for adult patients who received at least one vasopressor over 6 h within the first 24 h of intensive care unit(ICU) admission. Dynamic modulation of the MAP/NEQ index by the synergistic use of various low-dose vasopressors targeting urine output may be beneficial for exploring individualized optimization of MAP.

Efficacy of opioids for traumatic pain in the emergency department: a systematic review and Bayesian network meta-analysis.

Aim: To systematically assess and rank the efficacy of opioid medications for traumatic pain in the emergency department in terms of pain relief, adverse events and rescue analgesia. Methods: Four databases were systematically searched until 26 September 2022: PubMed, Embase, Cochrane Library, and Web of Science. Outcomes were pain relief, adverse events (dizziness, hypotension, pruritus, sedation), and rescue analgesia. For each outcome, network plots were drawn to exhibit direct and indirect comparisons, and rank probabilities were utilized to rank the efficacy of different opioids. Results: Twenty studies of 3,040 patients were eligible for this network meta-analysis. According to the rank probabilities, the top three analgesic medications for pain relief may be sufentanil (78.29% probability of ranking first), buprenorphine (48.54% probability of ranking second) and fentanyl (53.25% probability of ranking third); buprenorphine (31.20%), fentanyl (20.14%) and sufentanil (21.55%) were least likely to cause dizziness; the top three analgesic medications which were least likely to cause hypotension were buprenorphine (81.64%), morphine (45.02%) and sufentanil (17.27%); butorphanol (40.56%), morphine (41.11%) and fentanyl (14.63%) were least likely to cause pruritus; the top three medications which were least likely to cause sedation were hydrocodone + acetaminophen (97.92%), morphine (61.85%) and butorphanol (55.24%); patients who received oxycodone (83.64%), butorphanol (38.31%) and fentanyl (25.91%) were least likely to need rescue analgesia in sequence. Conclusion: Sufentanil, buprenorphine and fentanyl may be superior to other opioid medications in terms of pain relief and the incidence of dizziness, hypotension and pruritus, which might be selected as opioid analgesics for traumatic pain in the emergency setting.

1,3-Pentadiene-Assistant Living Anionic Terpolymerization: Composition Impact on Kinetics and Microstructure Sequence Primary Analysis.

The combination of a living anionic technology and a unique alternating strategy provided an exciting opportunity to prepare novel and well-defined poly(1,3-pentadiene-co-syrene-co-1,1-diphenylethylene) resins consisting of three alternating sequences of modules (A/B/C zwitterions). "A" being Styrene (St)/1,3-pentadiene (PD), "B" being diphenylethylene (DPE)/PD, "A" being DPE/St, respectively, A wide composition range of new polyolefin resins, i.e., poly (A-co-B), poly (A-co-C), and poly (B-co-C), with controlled molecular weight and very narrow molecular weight and composition distributions have been prepared by a one-pot living characteristic method. In the section of kinetic analysis, the terpolymer yields and kinetic parameters were strongly dependent on the feed/comonomer ratio as well as the content of the alternating structure. The competition copolymerization behaviors of A/B, B/C, and A/C were studied in detail in this work. By contrast, the microstructure and the thermal property of the resulting terpolymer were investigated via Nuclear magnetic resonance (NMR) and Differential scanning calorimetry (DSC) analysis. The results of 1H NMR tracking the change of [Aromatic ring]/[C=C] value indicated the distinctive copolymer-ization behavior of the selective "alternating-modules". The glass transition temperature (Tg) was very sensitive to the terpolymer composition. By contrast to poly(A-ran-B) with only one obvious Tg, there were two Tgs in the A/C and B/C copolymerization cases. Moreover, the desirable high Tg ~ 140 °C resin was limited to the terpolymers with up to 50 mol % DPE. Finally, the "ABC-X" mechanism was proposed to interpret the unique terpolymerization behavior, which belongs to the classical "bond-forming initiation" theory.

Superior Fidelity and Distinct Editing Outcomes of SaCas9 Compared to SpCas9 in Genome Editing.

A series of the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) systems have been engineered for genome editing. The most widely used Cas9 is SpCas9 from Streptococcus pyogenes and SaCas9 from Staphylococcus aureus. However, a comparison of their detailed gene editing outcomes is still lacking. By characterizing the editing outcomes of 11 sites in human induced pluripotent stem cells (iPSCs) and K562 cells, we found that SaCas9 could edit the genome with greater efficiency than SpCas9. We also compared the spacer lengths of single-guide RNA (sgRNA, 18-21 nt for SpCas9 and 19-23 nt for SaCas9) and found that the optimal spacer lengths were 20 nt and 21 nt for SpCas9 and SaCas9, respectively. However, the optimal spacer size for a particular guide RNA ranged from 18-21 nt or 21-22 nt for SpCas9 and SaCas9, respectively. Furthermore, SpCas9 exhibited a more substantial bias than SaCas9 for nonhomologous end-joining (NHEJ) +1 insertion at the fourth nucleotide upstream of the protospacer adjacent motif (PAM), characteristic of a staggered cut. Accordingly, editing with SaCas9 led to higher knock-in efficiencies of NHEJ-mediated double-stranded oligodeoxynucleotide (dsODN) insertion or adeno-associated virus serotype 6 (AAV6) donor-mediated homology-directed repair (HDR). Finally, GUIDE-seq analysis revealed that SaCas9 exhibited significantly reduced off-target effects compared with SpCas9. Our work indicates the superior performance of SaCas9 to SpCas9 in transgene integration-based therapeutic gene editing and the necessity to identify the optimal spacer length to achieve desired editing results.

Improved and Flexible HDR Editing by Targeting Introns in iPSCs.

Highly efficient gene knockout (KO) editing of CRISPR-Cas9 has been achieved in iPSCs, whereas homology-directed repair (HDR)-mediated precise gene knock-in (KI) and high-level expression are still bottlenecks for the clinical applications of iPSCs. Here, we developed a novel editing strategy that targets introns. By targeting the intron before the stop codon, this approach tolerates reading frameshift mutations caused by nonhomologous end-joining (NHEJ)-mediated indels, thereby maintaining gene integrity without damaging the non-HDR-edited allele. Furthermore, to increase the flexibility and screen for the best intron-targeting sgRNA, we designed an HDR donor with an artificial intron in place of the endogenous intron. The presence of artificial introns, particularly an intron that carries an enhancer element, significantly increased the reporter expression levels in iPSCs compared to the intron-deleted control. In addition, a combination of the small molecules M3814 and trichostatin A (TSA) significantly improves HDR efficiency by inhibiting NHEJ. These results should find applications in gene therapy and basic research, such as creating reporter cell lines.

Effective control of large deletions after double-strand breaks by homology-directed repair and dsODN insertion.

BACKGROUND: After repairing double-strand breaks (DSBs) caused by CRISPR-Cas9 cleavage, genomic damage, such as large deletions, may have pathogenic consequences. RESULTS: We show that large deletions are ubiquitous but are dependent on editing sites and cell types. Human primary T cells display more significant deletions than hematopoietic stem and progenitor cells (HSPCs), whereas we observe low levels in induced pluripotent stem cells (iPSCs). We find that the homology-directed repair (HDR) with single-stranded oligodeoxynucleotides (ssODNs) carrying short homology reduces the deletion damage by almost half, while adeno-associated virus (AAV) donors with long homology reduce large deletions by approximately 80%. In the absence of HDR, the insertion of a short double-stranded ODN by NHEJ reduces deletion indexes by about 60%. CONCLUSIONS: Timely bridging of broken ends by HDR and NHEJ vastly decreases the unintended consequences of dsDNA cleavage. These strategies can be harnessed in gene editing applications to attenuate unintended outcomes.

HDAC inhibitors improve CRISPR-mediated HDR editing efficiency in iPSCs.

Genome-edited human induced pluripotent stem cells (iPSCs) hold great promise for therapeutic applications. However, low editing efficiency has hampered the applications of CRISPR-Cas9 technology in creating knockout and homology-directed repair (HDR)-edited iPSC lines, particularly for silent genes. This is partially due to chromatin compaction, inevitably limiting Cas9 access to the target DNA. Among the six HDAC inhibitors we examined, vorinostat, or suberoylanilide hydroxamic acid (SAHA), led to the highest HDR efficiency at both open and closed loci, with acceptable toxicity. HDAC inhibitors equally increased non-homologous end joining (NHEJ) editing efficiencies (∼50%) at both open and closed loci, due to the considerable HDAC inhibitor-mediated increase in Cas9 and sgRNA expression. However, we observed more substantial HDR efficiency improvement at closed loci relative to open chromatin (2.8 vs. 1.7-fold change). These studies provide a new strategy for HDR-editing of silent genes in iPSCs.

Dynamics and competition of CRISPR-Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing.

Investigations of CRISPR gene knockout editing profiles have contributed to enhanced precision of editing outcomes. However, for homology-directed repair (HDR) in particular, the editing dynamics and patterns in clinically relevant cells, such as human iPSCs and primary T cells, are poorly understood. Here, we explore the editing dynamics and DNA repair profiles after the delivery of Cas9-guide RNA ribonucleoprotein (RNP) with or without the adeno-associated virus serotype 6 (AAV6) as HDR donors in four cell types. We show that editing profiles have distinct differences among cell lines. We also reveal the kinetics of HDR mediated by the AAV6 donor template. Quantification of T50 (time to reach half of the maximum editing frequency) indicates that short indels (especially +A/T) occur faster than longer (>2 bp) deletions, while the kinetics of HDR falls between NHEJ (non-homologous end-joining) and MMEJ (microhomology-mediated end-joining). As such, AAV6-mediated HDR effectively outcompetes the longer MMEJ-mediated deletions but not NHEJ-mediated indels. Notably, a combination of small molecular compounds M3814 and Trichostatin A (TSA), which potently inhibits predominant NHEJ repairs, leads to a 3-fold increase in HDR efficiency.

Liver Kinase B1 Fine-Tunes Lineage Commitment of Human Fetal Synovium-Derived Stem Cells.

Liver kinase B1 (LKB1), a serine/threonine protein, is a key regulator in stem cell function and energy metabolism. Herein, we describe the role of LKB1 in modulating the differentiation of synovium-derived stem cells (SDSCs) toward chondrogenic, adipogenic, and osteogenic lineages. Human fetal SDSCs were transduced with CRISPR associated protein 9 (Cas9)-single-guide RNA vectors to knockout or lentiviral vectors to overexpress the LKB1 gene. Analyses including ICE (Inference of CRISPR Edits) data from Sanger sequencing and quantitative polymerase chain reaction (qPCR) as well as Western blot demonstrated successful knockout (KO) or overexpression (OE) of LKB1 in human fetal SDSCs without any detectable side effects in morphology, proliferation rate, and cell cycle. LKB1 KO increased CD146 expression; interestingly, LKB1 OE increased SSEA4 level. The qPCR data showed that LKB1 KO upregulated the levels of SOX2 and NANOG while LKB1 OE lowered the expression of POU5F1 and KLF4. Furthermore, LKB1 KO enhanced, and LKB1 OE inhibited, chondrogenic and adipogenic differentiation potential. However, perhaps due to the inherent inability to achieve osteogenesis, LKB1 did not obviously affect osteogenic differentiation. These data demonstrate that LKB1 plays a significant role in determining human SDSCs' adipogenic and chondrogenic differentiation, which might provide an approach for fine-tuning the direction of stem cell differentiation in tissue engineering and regeneration. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:258-268, 2020.

Unique anabolic action of stem cell gene therapy overexpressing PDGFB-DSS6 fusion protein in OVX osteoporosis mouse model.

In the present study we sought to improve the efficacy and safety of our Sca1+ PDGFB stem cell gene therapy for osteoporosis in ovariectomized (OVX) mouse model. This therapy is administered by marrow transplantation. We established the promise of this approach by previously showing that this therapy in normal mice increase bone density, increased endosteal cortical and trabecular bone formation, caused de novo trabecular bone formation, increased cortical thickness and improve bone strength. In the current study we produced a fusion gene, PDGFB-DSS6. We reasoned that the DSS6, calcium binding protein would trap the PDGFB at the bone surface and thereby limit the amount of PDGFB required to produce an optimal bone formation response, i.e. efficacy with a lower engraftment. The result shows that indeed with a very low level of engraftment we achieved a large increase in bone formation in the OVX model of bone loss. Serum analysis for biochemical marker of new bone formation showed an approximate 75% increase in alkaline phosphatase levels in Sca1+PDGFB-DSS6 group as compared to other groups. Quantitative analysis of bone by microCT showed a massive increase in trabecular bone density and trabecular connectivity of the femur in the metaphysis in Sca1+ PDGFB-DSS6 group. The increased cortical porosity produced by OVX was replaced by the Sca1+ PDGFB-DSS6 therapy but not by the positive control Sca1+ PDGFB. Additionally, an increase in the femur bone strength was also observed specifically in Sca1+ PDGFB-DSS6 as compared to other treatment groups, emphasizing the functional significance of the observed anabolic action is on bone formation. In future work we will focus on nontoxic preconditioning of our marrow transplantation procedure and also on transcriptional control of therapeutic gene expression to avoid excess bone formation.

Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in the mouse.

BACKGROUND: Hemophilia A, a bleeding disorder resulting from F8 mutations, can only be cured by gene therapy. A promising strategy is CRISPR-Cas9-mediated precise insertion of F8 in hepatocytes at highly expressed gene loci, such as albumin (Alb). Unfortunately, the precise in vivo integration efficiency of a long insert is very low (~ 0.1%). RESULTS: We report that the use of a double-cut donor leads to a 10- to 20-fold increase in liver editing efficiency, thereby completely reconstituting serum F8 activity in a mouse model of hemophilia A after hydrodynamic injection of Cas9-sgAlb and B domain-deleted (BDD) F8 donor plasmids. We find that the integration of a double-cut donor at the Alb locus in mouse liver is mainly through non-homologous end joining (NHEJ)-mediated knock-in. We then target BDDF8 to multiple sites on introns 11 and 13 and find that NHEJ-mediated insertion of BDDF8 restores hemostasis. Finally, using 3 AAV8 vectors to deliver genome editing components, including Cas9, sgRNA, and BDDF8 donor, we observe the same therapeutic effects. A follow-up of 100 mice over 1 year shows no adverse effects. CONCLUSIONS: These findings lay the foundation for curing hemophilia A by NHEJ knock-in of BDDF8 at Alb introns after AAV-mediated delivery of editing components.

Impact of Fibronectin Knockout on Proliferation and Differentiation of Human Infrapatellar Fat Pad-Derived Stem Cells.

Fibronectin plays an essential role in tissue development and regeneration. However, the effects of fibronectin knockout (FN1-KO) on stem cells' proliferation and differentiation remain unknown. In this study, CRISPR/Cas9 generated FN1-KO in human infrapatellar fat pad-derived stem cells (IPFSCs) was evaluated for proliferation ability including cell cycle and surface markers as well as stemness gene expression and for differentiation capacity including chondrogenic and adipogenic differentiation. High passage IPFSCs were also evaluated for proliferation and differentiation capacity after expansion on decellularized ECM (dECM) deposited by FN1-KO cells. Successful FN1-KO in IPFSCs was confirmed by Sanger sequencing and Inference of CRISPR Edits analysis (ICE) as well as immunostaining for fibronectin expression. Compared to the GFP control, FN1-KO cells showed an increase in cell growth, percentage of cells in the S and G2 phases, and CD105 and CD146 expression but a decrease in expression of stemness markers CD73, CD90, SSEA4, and mesenchymal condensation marker CDH2 gene. FN1-KO decreased both chondrogenic and adipogenic differentiation capacity. Interestingly, IPFSCs grown on dECMs deposited by FN1-KO cells exhibited a decrease in cell proliferation along with a decline in CDH2 expression. After induction, IPFSCs plated on dECMs deposited by FN1-KO cells also displayed decreased expression of both chondrogenic and adipogenic capacity. We concluded that FN1-KO increased human IPFSCs' proliferation capacity; however, this capacity was reversed after expansion on dECM deposited by FN1-KO cells. Significance of fibronectin in chondrogenic and adipogenic differentiation was demonstrated in both FN1-KO IPFSCs and FN(-) matrix microenvironment.

Highly efficient genome editing via CRISPR-Cas9 in human pluripotent stem cells is achieved by transient BCL-XL overexpression.

Genome editing of human induced pluripotent stem cells (iPSCs) is instrumental for functional genomics, disease modeling, and regenerative medicine. However, low editing efficiency has hampered the applications of CRISPR-Cas9 technology in creating knockin (KI) or knockout (KO) iPSC lines, which is largely due to massive cell death after electroporation with editing plasmids. Here, we report that the transient delivery of BCL-XL increases iPSC survival by ∼10-fold after plasmid transfection, leading to a 20- to 100-fold increase in homology-directed repair (HDR) KI efficiency and a 5-fold increase in non-homologous end joining (NHEJ) KO efficiency. Treatment with a BCL inhibitor ABT-263 further improves HDR efficiency by 70% and KO efficiency by 40%. The increased genome editing efficiency is attributed to higher expressions of Cas9 and sgRNA in surviving cells after electroporation. HDR or NHEJ efficiency reaches 95% with dual editing followed by selection of cells with HDR insertion of a selective gene. Moreover, KO efficiency of 100% can be achieved in a bulk population of cells with biallelic HDR KO followed by double selection, abrogating the necessity for single cell cloning. Taken together, these simple yet highly efficient editing strategies provide useful tools for applications ranging from manipulating human iPSC genomes to creating gene-modified animal models.

High-Level Precise Knockin of iPSCs by Simultaneous Reprogramming and Genome Editing of Human Peripheral Blood Mononuclear Cells.

We have developed an improved episomal vector system for efficient generation of integration-free induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells. More recently, we reported that the use of an optimized CRISPR-Cas9 system together with a double-cut donor increases homology-directed repair-mediated precise gene knockin efficiency by 5- to 10-fold. Here, we report the integration of blood cell reprogramming and genome editing in a single step. We found that expression of Cas9 and KLF4 using a single vector significantly increases genome editing efficiency, and addition of SV40LT further enhances knockin efficiency. After these optimizations, genome editing efficiency of up to 40% in the bulk iPSC population can be achieved without any selection. Most of the edited cells show characteristics of iPSCs and genome integrity. Our improved approach, which integrates reprogramming and genome editing, should expedite both basic research and clinical applications of precision and regenerative medicine.

HOXA4, down-regulated in lung cancer, inhibits the growth, motility and invasion of lung cancer cells.

The involvement of HOXA4 in colorectal cancer and epithelial ovarian cancer has been reported. Although it has been reported that the Hoxa4 gene is involved in the patterning of the mouse lung during embryonic development, little is known about the biological functions of HOXA4 in lung cancer. In the current study, HOXA4 expression was down-regulated in lung cancer tissues when compared with non-cancerous tissues. HOXA4 expression was associated with tumor size, TNM stage, lymph node metastasis and prognosis. Bioinformatics analysis revealed that HOXA4 expression was negatively correlated with cell cycle, metastasis, and the Wnt signaling pathway. Moreover, HOXA4 overexpression in lung cancer cell lines suppressed cell proliferation, migration, and invasion. HOXA4 decreased the protein expression levels of ß-catenin, Cyclin D1, c-Myc and Survivin, indicating the inhibition of Wnt signaling. HOXA4 significantly increased the protein and mRNA levels of glycogen synthase kinase-3ß (GSK3ß) by promoting its transcription. Furthermore, inhibition of GSK3ß by LiCl abolished the suppression of cell growth, migration, and invasion mediated by HOXA4. Overexpression of HOXA4 in xenograft tumors also decreased tumor growth and Wnt signaling. Collectively, these data suggest that HOXA4 is a potential diagnostic and prognostic marker in lung cancer, and its overexpression could inhibit lung cancer progression in part by promoting GSK3ß transcription.

Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage.

BACKGROUND: Precise genome editing via homology-directed repair (HDR) after double-stranded DNA (dsDNA) cleavage facilitates functional genomic research and holds promise for gene therapy. However, HDR efficiency remains low in some cell types, including some of great research and clinical interest, such as human induced pluripotent stem cells (iPSCs). RESULTS: Here, we show that a double cut HDR donor, which is flanked by single guide RNA (sgRNA)-PAM sequences and is released after CRISPR/Cas9 cleavage, increases HDR efficiency by twofold to fivefold relative to circular plasmid donors at one genomic locus in 293 T cells and two distinct genomic loci in iPSCs. We find that a 600 bp homology in both arms leads to high-level genome knockin, with 97-100% of the donor insertion events being mediated by HDR. The combined use of CCND1, a cyclin that functions in G1/S transition, and nocodazole, a G2/M phase synchronizer, doubles HDR efficiency to up to 30% in iPSCs. CONCLUSIONS: Taken together, these findings provide guidance for the design of HDR donor vectors and the selection of HDR-enhancing factors for applications in genome research and precision medicine.

[Influence of chronic lead exposure in rats during the developmental stage on expression of leptin in plasma, cerebrospinal fluid, and hippocampus].

OBJECTIVE: To investigate the influence of lead exposure in rats during the developmental stage on the expression of leptin in plasma, cerebrospinal fluid, and hippocampus, as well as investigating whether leptin is associated with the mechanism of cognitive impairment induced by lead exposure. METHODS: The rat model of cognitive impairment after chronic lead exposure was established by adding lead acetate into drinking water. According to the concentration of lead acetate in drinking water, the rats were divided into control (0 ppm), low-lead (50 ppm), medium-lead (200 ppm), and high-lead groups (1 000 ppm), with 16 rats in each group. Atomic absorption spectrometry was used to measure the content of lead in the plasma, cerebrospinal fluid and hippocampus. ELISA was used to measure the level of leptin in the plasma and cerebrospinal fluid. Immunohistochemistry was used to observe the distribution of leptin protein in the hippocampus. Western blot was used for relative quantification of leptin proteins in the hippocampus. RESULTS: Compared with the control group, the lead exposure groups showed significant increases in the content of lead in blood, cerebrospinal fluid, and hippocampus (P<0.01), as well as significant reductions in the levels of leptin in plasma and cerebrospinal fluid (P<0.05). The results of immunohistochemical staining showed that leptin was mainly distributed in the cytoplasm of pyramidal neurons in the hippocampal CA region. The results of Western blot showed that compared with the control group, the three lead exposure groups showed a slight increase in the protein expression of leptin in the hippocampus (P>0.05). CONCLUSIONS: Lead exposure can reduce the levels of leptin in plasma and cerebrospinal fluid in rats, which may be associated with the mechanism of cognitive impairment induced by lead exposure.

Leptin and IL-8: two novel cytokines screened out in childhood lead exposure.

Lead is a toxic heavy metal with many recognized adverse health side effects. The central nervous system is the main target of lead toxicity. Although many studies on lead toxicity were conducted, the mechanism of lead toxicity remains uncertain. One possible attribution is the immature blood-brain barrier that causes lead exposure in children. Few studies have investigated the cytokine changes caused by this exposure. Novel cytokines were detected by RayBio(®) Human Cytokine Antibody Array and validated by enzyme-linked immunosorbent assay. Several children were admitted to West China Second University Hospital, after a serious lead pollution event in longchang, Sichuan, China. A total of 4 children with elevated blood lead levels (BLLs) and 4 children with low BLLs were randomly chosen in the discovery set, and 40 children with elevated BLLs and 40 children with low BLLs were included in the validation set. Leptin and interleukin-8 (IL-8) were identified to be significantly different between children with elevated and low BLLs via RayBio(®) Human Cytokine Antibody Array. In the validation set, IL-8 was higher in children with elevated BLLs [median(P25-P75), 117.69(52.31-233.63) pg/mL] than in children with low BLLs [median(P25-P75): 17.70(10.75-26.52) pg/mL] (p=0.000). Leptin was lower in children with elevated BLLs [median(P25-P75): 1658.23(1421.86-2606.55) pg/mL] than in children with low BLLs [median(P25-P75): 4168.68(3246.32-4744.94) pg/mL] (p=0.000). In children with low BLLs, leptin was higher in children with BLLs<3 µg/dL (N=7) [median(P25-P75): 7220.86(4265.72-7555.15) pg/mL] than in children with BLL ≥ 3 µg/dL (N=33) [median(P25-P75): 4103.86(3163.40-4678.34) pg/mL] (p=0.026); IL-8 was significantly different in children with BLL<4 µg/dL (N=13) [median(P25-P75): 12.49(8.25-14.86) pg/mL] than in children with BLL≥4 µg/dL (N=27) [median(P25-P75): 21.98(13.64-33.50) pg/mL] (p=0.013). The results defined specific changes in cytokine expressions to lead exposure, which can be used to explore the mechanism of lead toxicity and monitor lead exposure.

Validity and reliability of the Taiwanese version of the Pittsburgh Sleep Quality Index in cancer patients.

BACKGROUND: Sleep disturbance remains a common symptom among cancer patients. Assessment of sleep disturbance in cancer patients is hindered by infrequent use of standardized tools for sleep management. OBJECTIVES: The purposes of this study were to validate the Taiwanese version of the Pittsburgh Sleep Quality Index (PSQI-T) and to determine detection cut-off points in cancer patients. DESIGN: A cross-sectional and descriptive correlational design. PARTICIPANTS: A sample of 205 Taiwanese patients with various cancer diagnoses. METHODS: The survey included the Pittsburgh Sleep Quality Index-Taiwanese version, the MD Anderson Symptom Inventory-Taiwanese version, the Brief Fatigue Inventory-Taiwanese version, the seven-day sleep log, and Manual of Mental Disorders, Fourth Edition. RESULTS: The internal consistency Cronbach's alpha for the PSQI was 0.79. Test-retest reliability was 0.91 for the global score over a 20- to 28-day interval in a sample of 16 patients. Construct validity was established by a significant relationship of the PSQI-T global score to the total symptom severity score and the fatigue severity score. Convergent validity was examined by correlating the PSQI-T scores and scores of the DSM-IV and scores on the seven-day sleep log. Known-group validity was established by comparing PSQI-T scores for patients having low fatigue levels and those having high fatigue levels. Receiver-operating characteristic (ROC) curves were used to determine the detection cut-off points. CONCLUSIONS: We found that a PSQI-T global score of 8 generates the best sensitivity and specificity for measuring sleep disturbance in cancer patients. The PSQI-T is a reliable, valid, and sensitive instrument for measuring sleep quality among Taiwanese cancer patients.