The role of tumor-associated macrophages in glioma cohort: through both traditional RNA sequencing and single cell RNA sequencing.

Gliomas are the leading cause in more than 50% of malignant brain tumor cases. Prognoses, recurrences, and mortality are usually poor for gliomas that have malignant features. In gliomas, there are four grades, with grade IV gliomas known as glioblastomas (GBM). Currently, the primary methods employed for glioma treatment include surgical removal, followed by chemotherapy after the operation, and targeted therapy. However, the outcomes of these treatments are unsatisfactory. Gliomas have a high number of tumor-associated macrophages (TAM), which consist of brain microglia and macrophages, making them the predominant cell group in the tumor microenvironment (TME). The glioma cohort was analyzed using single-cell RNA sequencing to quantify the genes related to TAMs in this study. Furthermore, the ssGSEA analysis was utilized to assess the TAM-associated score in the glioma group. In the glioma cohort, we have successfully developed a prognostic model consisting of 12 genes, which is derived from the TAM-associated genes. The glioma cohort demonstrated the predictive significance of the TAM-based risk model through survival analysis and time-dependent ROC curve. Furthermore, the correlation analysis revealed the significance of the TAM-based risk model in the application of immunotherapy for individuals diagnosed with GBM. Ultimately, the additional examination unveiled the prognostic significance of PTX3 in the glioma group, establishing it as the utmost valuable prognostic indicator in patients with GBM. The PCR assay revealed the PTX3 is significantly up-regulated in GBM cohort. Additionally, the assessment of cell growth further confirms the involvement of PTX3 in the GBM group. The analysis of cell proliferation showed that the increased expression of PTX3 enhanced the ability of glioma cells to proliferate. The prognosis of glioblastomas and glioma is influenced by the proliferation of tumor-associated macrophages.

TPPP3 promote epithelial-mesenchymal transition via Snail1 in glioblastoma.

Tubulin polymerization promoting protein 3 (TPPP3), a member of the tubulin polymerization family, participates in cell progressions in several human cancers, its biological function and the underlying mechanisms in glioblastoma multiforme (GBM) remain unclear. Here, we investigated the role and application value of TPPP3 in gliomas and found that the expression of TPPP3 in glioma was higher than that in normal brain tissue (NBT), and increased with the grade of glioma. Up-regulation of TPPP3 expression in glioblastoma cells confer stronger ability of migration, invasion, proliferation and lower apoptosis in vitro. Inhibition of TPPP3 expression in GBM could reduce the migration, invasion, proliferation and induce the apoptosis of glioblastoma cells. TPPP3 affected the process of EMT by regulating the expression of Snail 1 protein. In clinical data analysis, we found a positive correlation between TPPP3 and Snail1 protein expression levels in glioblastomas. Low TPPP3 expression leads to better survival expectations in glioblastomas patients. The content of this study paves the way for further in-depth exploration of the role of TPPP3 in glioblastoma in the future, and provides new treatment and research directions.

PRL1 Promotes Glioblastoma Invasion and Tumorigenesis via Activating USP36-Mediated Snail2 Deubiquitination.

Regenerating liver phosphatase 1 (PRL1) is an established oncogene in various cancers, although its biological function and the underlying mechanisms in glioblastoma multiforme (GBM) remain unclear. Here, we showed that PRL1 was significantly upregulated in glioma tissues and cell lines, and positively correlated with the tumor grade. Consistently, ectopic expression of PRL1 in glioma cell lines significantly enhanced their tumorigenicity and invasion both in vitro and in vivo by promoting epithelial-mesenchymal transition (EMT). Conversely, knocking down PRL1 blocked EMT in GBM cells, and inhibited their invasion, migration and tumorigenic growth. Additionally, PRL1 also stabilized Snail2 through its deubiquitination by activating USP36, thus revealing Snail2 as a crucial mediator of the oncogenic effects of PRL1 in GBM pathogenesis. Finally, PRL1 protein levels were positively correlated with that of Snail2 and predicted poor outcome of GBMs. Collectively, our data support that PRL1 promotes GBM progression by activating USP36-mediated Snail2 deubiquitination. This novel PRL1/USP36/Snail2 axis may be a promising therapeutic target for glioblastoma.

Ancient Genomes Reveal the Evolutionary History and Origin of Cashmere-Producing Goats in China.

Goats are one of the most widespread farmed animals across the world; however, their migration route to East Asia and local evolutionary history remain poorly understood. Here, we sequenced 27 ancient Chinese goat genomes dating from the Late Neolithic period to the Iron Age. We found close genetic affinities between ancient and modern Chinese goats, demonstrating their genetic continuity. We found that Chinese goats originated from the eastern regions around the Fertile Crescent, and we estimated that the ancestors of Chinese goats diverged from this population in the Chalcolithic period. Modern Chinese goats were divided into a northern and a southern group, coinciding with the most prominent climatic division in China, and two genes related to hair follicle development, FGF5 and EDA2R, were highly divergent between these populations. We identified a likely causal de novo deletion near FGF5 in northern Chinese goats that increased to high frequency over time, whereas EDA2R harbored standing variation dating to the Neolithic. Our findings add to our understanding of the genetic composition and local evolutionary process of Chinese goats.

An LC-MS/MS assay to determine plasma pharmacokinetics of cyclic thymic hexapeptide (cTP6) in rhesus monkeys.

A robust and simple method for absolute quantification of a novel bidirectional immunomodulatory drug candidate, cyclic thymic hexapeptide (cTP6), in rhesus monkey plasma was developed and validated by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Plasma proteins were precipitated by adding four volumes of acetonitrile. Peptides in the supernatant were separated by liquid chromatography on an Agilent Zorbax Eclipse Plus-C18 chromatographic column with gradient elution using 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in methanol (mobile phase B) at 0.2 mL/min. The analytes were identified by triple quadrupole mass spectrometry in positive ion-mode. The assay was linear over a concentration range of 10-5000 ng/mL for cTP6, with a lower limit of quantification (LLOQ) of 10 ng/mL. Intra- and inter-day precision of the assay at three concentrations were 1.51-7.70% with accuracy of 95.1-104.2%. The average recovery of cTP6 for three concentration levels was 59.6-64.0%. No significant matrix effect was observed. Peptide cTP6 was detected in plasma of live rhesus monkeys up to 6-8h after intra-muscular injection. The half-life was 2.24-2.95 h. The result revealed a nonlinear pharmacokinetic response to increasing doses of cTP6 (100, 200, 500 µg/kg). For the multiple dose study of cTP6, the drug did not accumulate during daily administration at 100 µg/kg for 7 consecutive days in rhesus monkeys.

Qualitative and quantitative studies on human B7.1-Fc fusion protein and the application in pharmacokinetic study in rhesus monkeys.

A sensitive, accurate, and precise enzyme immunoassay (EIA) for the quantification of intact human B7.1-Fc in rhesus monkey serum was validated, and the characteristics of B7.1 and Fc moiety of fusion protein were identified by surface plasmon resonance (SPR) and flow-cytometric method, respectively. B7.1-Fc bound to CD28 and CTLA-4 with K(d) values of 45.1 and 9.58 nM, respectively, which were very closed to the previous reports and the function of Fc moiety of fusion protein was also confirmed by Fc receptor binding assay and IL-8 releasing assay. To monitor the intact protein, the EIA method employed a sandwich scheme in which a multiclonal anti-human IgG (Fc specific) antibody and a monoclonal anti-human B7.1 antibody were served as capture and detection antibody, respectively. This EIA has a range of reliable response of 0.5-32 ng/ml. The LLOQ was established at 0.5 ng/ml. The intra-assay precision and accuracy were 6.1-8.8% and (3.0-9.0)%, respectively with the inter-assay precision and accuracy were 5.7-11.5% and (10.7-9.1)%, respectively. Stability was established under certain conditions and no significant differences were found. This validated EIA assay was then successfully employed in the assessment of pharmacokinetic behavior of B7.1-Fc in rhesus monkeys after intravenous infusion, and a non-linear characteristics was established across the investigated dosage range (32-320 µg/kg).

Quantitative analysis of a novel HIV fusion inhibitor (sifuvirtide) in HIV infected human plasma using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry.

A sensitive method for measuring sifuvirtide, a novel HIV fusion inhibitor peptide drug in HIV-1(+) human plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. The plasma samples were treated by solvent/detergent (S/D) method to inactivate viral activity before analysis. After protein precipitation sifuvirtide was determined by LC-MS/MS. A structure analog was used as internal standard (IS). The mass spectrometer was operated in positive ion and multiple reaction monitoring mode with transitions m/z 946.3-->159.0 for sifuvirtide and 951.7-->159.2 for IS. The intra-day precision ranged from 2.74% to 7.57% with accuracy from 91.63% to 102.53%. The inter-day precision ranged from 2.65% to 3.58% and the accuracy from 95.53% to 105.28%. Stability studies showed that sifuvirtide was stable both during the assay procedure and long-term storage. The lower limit of quantitation (LLOQ) was 9.75ngml(-1). The method was used for analyzing samples from phase IIa clinical study of sifuvirtide in China.

The extract of inflamed rabbit skin induced by inoculation of vaccinia virus possesses antioxidant and neuroprotective effects in acute ischemic stroke.

Acute cerebral ischemia remains a major cause for death and disability but current therapeutic options are limited. A mixture of biological agents extracted from the inflamed rabbit skin induced by inoculation of vaccinia virus has been shown to reduce ischemia-induced cerebral edema in vivo. In the current study we show that treatment with such a mixture can also significantly reduce the infarct volume and ameliorate the neurologic deficits in animals after acute occlusion of the middle cerebral artery. Further studies demonstrate that this mixture possesses antioxidant capacities that can decrease the levels of lactic acidosis and increase the activities of superoxide dismutase in the lesional brain. It can also preserve the viability of neuronal cells under local hypoxia and hypoglycemia environments or after exposure to hydrogen peroxide in vitro. Such extract, therefore, may become a potential treatment regimen with promising therapeutic value in human subjects.

Development of a rapid and sensitive LC-MS/MS assay for the determination of combretastatin A4 phosphate, combretastatin A4 and combretastatin A4 glucuronide in beagle dog plasma and its application to a pharmacokinetic study.

This study details the development and validation of a simple and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the quantification of combretastatin A-4 3-O-phosphate (CA4P), combretastatin A4 (CA4) and its main metabolite, combretastatin A4 glucuronide (CA4G), in beagle dog plasma. Sample pretreatment includes simple protein precipitation by adding methanol to the plasma sample containing an internal standard (colchicine). LC separation was successfully accomplished on a Waters RP8 Symmetryshield column (3.0mmx150mm, i.d., 5microm) with a gradient mobile phase of methanol (0.1% formic acid, v/v) and water (20mM ammonium acetate) at a flow rate 0.8mLmin(-1). The three analytes were detected in the positive/negative ion alternate mode, negative ion mode for CA4G and positive ion mode for CA4P and CA4. Multiple reaction monitoring (MRM) was used for determination of three analytes. Calibration curves were linear in the concentration range of 5-5000ngmL(-1) for CA4P (r>or=0.999), 5-3000ngmL(-1) for CA4 (r>or=0.999) and 5-5000ngmL(-1) for CA4G (r>or=0.999). All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. The method was reliable and has been successfully applied to a pharmacokinetic study of CA4P in beagle dogs via intravenous drop infusion at dose rates of 1, 3 and 9mgkg(-1). After daily intravenous drop infusions at 1mgkg(-1) for 7 consecutive days, the accumulation ratio was approximately 1.0, indicating no accumulation from multiple doses in beagles.

Development and application of a real-time PCR method for pharmacokinetic and biodistribution studies of recombinant adenovirus.

A replication-deficient recombinant adenovirus (Ad5-LFA-3/IgG(1)) that encodes secreted LFA-3/IgG(1) was constructed for gene therapy treatment of psoriasis. The purpose of this study was to develop a real-time PCR method for pharmacokinetic and biodistribution studies of Ad5-LFA-3/IgG(1) within the circulation and organs. This method showed good specificity, sensitivity and reproducibility over a wide dynamic range of concentrations. Quantitative measurement of recombinant adenoviral DNA suggested that the level of Ad5-LFA-3/IgG(1) DNA in circulating blood peaked within 10 min following intravenous injection in rhesus macaques. Following this peak, the adenoviral DNA level dropped significantly to a very low level. Real-time PCR revealed that Ad5-LFA-3/IgG(1) DNA was enriched in the spleen, lung and liver after injection of the adenovirus into rats through the tail vein. The adenoviral DNA was barely detected in other tissues. These data provide important information for clinical trials of Ad5-LFA-3/IgG(1) and confirm the utility of the real-time PCR assay for monitoring gene therapy trials.

A new approach for pharmacokinetics of single-dose cetuximab in rhesus monkeys by surface plasmon resonance biosensor.

A novel assay method has been developed and validated, using surface plasmon resonance (SPR), for quantitation of cetuximab (C225) in monkey serum. By injecting non-labeled antibody samples onto a biosensor surface on which epidermal growth factor receptor (EGFR) was immobilized, the concentration of C225 can be accurately measured. This assay has a range of reliable response from 0.05 to 50 microg/ml C225 in monkey serum, which was well fitted with a sigmoidal model. The immobilized EGFR was found to be stable for at least 100 regeneration cycles at room temperature. Intra- and inter-assay CVs ranged from 3.20% to 8.89% and from 5.93% to 11.11%, accuracy from 92% to 107.52% and from 90% to 106.88%, respectively. Matrices such as 50% human serum, 50% Sprague Dawley rat serum, chimeric recombinant anti-CD20 monoclonal antibody, human gamma-globulin and chimeric recombinant her2 antibody did not interfere with C225 analysis on the sensor surface. This is the first report on the quantitation of C225 in monkey serum by an optical biosensor technology. This method was used to characterize the pharmacokinetics of C225 in rhesus monkeys. After a single-dose of intravenous infusion administration of 7.5, 24 and 75 microg/kg, average C(max) ranged from 168+/-28 to 1624+/-113 microg/ml, and AUC(0-infinity) ranged from 15,739+/-1059 to 295,017+/-44,533 microg h/ml. C225 elimination followed a bi-exponential profile with t(1/2) ranging from 2.7+/-0.7 to 6.7+/-0.1 h. It was non-linear serum pharmacokinetics of C225 across the investigated dosage range in monkeys (7.5-75 mg/kg).

Validation of a sensitive gas chromatographic-mass spectrometric method for the simultaneous determination of beta-elemene and beta-elemenal in human plasma.

A sensitive gas chromatographic-mass spectrometric assay was described for determination of beta-elemene and beta-elemenal in human plasma, which has been successfully applied in clinical trial. After liquid-liquid extraction and gas chromatographic separation, the analytes were identified and quantitated. Calibration curves were linear in range from 31.25 to 8000 ng mL(-1) and the limit of quantification for both was 31.25 ng mL(-1). Intra- and inter-day precision at three concentrations were 2.3-8.3% with accuracy of -8.5 to 6.1% for elemene and 3.0-9.9% with accuracy of -2.3 to 5.9% for elemenal. The method was validated to be suitable for further pharmacokinetic study.

Single- and multiple-dose pharmacokinetics of exendin-4 in rhesus monkeys.

A radioimmunoassay (RIA) for the measurement of exendin-4 concentration in rhesus monkeys serum was developed and validated. The radioimmunoassay described here was sensitive, linear, accurate, precise, and reproducible. Range of the assay was 25-2000 pg/ml. Using this method we characterized the pharmacokinetics and accumulation of exendin-4 in rhesus monkeys. Following s.c. administration at doses rate of 1, 3 and 10 microg/kg, average C(max) ranged from 2.26+/-0.15 to 22.72+/-1.54 ng/ml, and AUC(0-infinity) ranged from 3.43+/-0.05 to 47.1+/-0.10 ng h/ml. As compared to the i.v. administration at a single dose of 3 microg/kg, the absolute bioavailability after s.c. administration were estimated to be 67.3+/-5.3, 75.1+/-4.7 and 72.7+/-8.4% for 1, 3 and 10 microg/kg dose, respectively. After daily s.c. administration at 10 microg/kg for 7 consecutive days, the accumulation ratio was approximately to 1.0, indicating no accumulation upon multiple doses in the monkeys.

Validation of a sensitive LC/MS/MS method for simultaneous quantitation of flupentixol and melitracene in human plasma.

A sensitive method has been developed and validated, using LC/ESI-MS/MS, for simultaneous quantitation of flupentixol and melitracen--antidepressant drugs, in human plasma. The quantitation of the target compounds was determined in a positive ion mode and multiple reaction monitoring (MRM). The method involved a repeated liquid-liquid extraction with diethyl ether and analytes were chromatographed on a C(8) chromatographic column by elution with acetonitrile-water-formic acid (36:64:1, v/v/v) and analyzed by tandem mass spectrometry. The method was validated over the concentration ranges of 26.1-2090 pg/ml for flupentixol and 0.206-4120 ng/ml for melitracen. The correlation coefficients of both analyst were >0.998 for six sets of calibration curves. The recovery was 60.9-75.1% for flupentixol, melitracen and internal standard. The lower limit of quantitation (LLOQ) detection was 26.1 pg/ml for flupentixol and 0.206 ng/ml for melitracen. Intra- and inter-day precision of the assay at three concentrations were 2.15-5.92% with accuracy of 97.6-103.0% for flupentixol and 0.5-6.36% with accuracy of 98.7-101.7% for melitracen. Stability of compounds was established in a battery of stability studies, i.e., bench-top, autosampler and long-term storage stability as well as freeze/thaw cycles. The method proved to be suitable for bioequivalence study of flupentixol and melitracen in healthy human male volunteers.