Machine learning identifies novel coagulation genes as diagnostic and immunological biomarkers in ischemic stroke.

BACKGROUND: Coagulation system is currently known associated with the development of ischemic stroke (IS). Thus, the current study is designed to identify diagnostic value of coagulation genes (CGs) in IS and to explore their role in the immune microenvironment of IS. METHODS: Aberrant expressed CGs in IS were input into unsupervised consensus clustering to classify IS subtypes. Meanwhile, key CGs involved in IS were further selected by weighted gene co-expression network analysis (WGCNA) and machine learning methods, including random forest (RF), support vector machine (SVM), generalized linear model (GLM) and extreme-gradient boosting (XGB). The diagnostic performance of key CGs were evaluated by receiver operating characteristic (ROC) curves. At last, quantitative PCR (qPCR) was performed to validate the expressions of key CGs in IS. RESULTS: IS patients were classified into two subtypes with different immune microenvironments by aberrant expressed CGs. Further WGCNA, machine learning methods and ROC curves identified ACTN1, F5, TLN1, JMJD1C and WAS as potential diagnostic biomarkers of IS. In addition, their expressions were significantly correlated with macrophages, neutrophils and/or T cells. GSEA also revealed that those biomarkers may regulate IS via immune and inflammation. Moreover, qPCR verified the expressions of ACTN1, F5 and JMJD1C in IS. CONCLUSIONS: The current study identified ACTN1, F5 and JMJD1C as novel coagulation-related biomarkers associated with IS immune microenvironment, which enriches our knowledge of coagulation-mediated pathogenesis of IS and sheds light on next-step in vivo and in vitro experiments to elucidate the relevant molecular mechanisms.

Cascade signal amplification strategy for the electrochemical aptasensing of nucleic acid: Combination of dual-output toehold-mediated DNA strand displacement, DNA walker and Exo III.

BACKGROUND: Sensitive and selective analysis of low content nucleic acid sequences plays an important role in pathogen analysis, disease diagnosis and biomedicine. The electrochemical biosensor based on toehold-mediated strand displacement reaction (TMSD) is highly attractive in nucleic acid detection due to their improved sensitivity and rapid response. But the traditional TMSD carried out on the electrode always with low displacement efficiency and complicated electrode operation, resulting in compromised sensing performance. There is a great need to construct a novel TMSD based electrochemical detection strategy to overcome such challenges in nucleic acid detecting. RESULT: Herein, a triple signal amplification electrochemical aptasensor was developed for ultrasensitive detection of CYFRA21-1 DNA. The dual-output toehold mediated strand displacement reaction (dTMSD) can convert one input to two strands output within one strand displacement cycle. So that it possesses a higher efficiency for improving the sensitivity in comparison with the single-output TMSD. And the fuel strand was configured with a tail to realize successive DNA circuits through self-propelling as a DNA walker. All the above processes were carried out on magnetic beads, which is conducive to achieving effective sample purification and minimizing the background signals. Besides, Exonuclease III was further amplified signal. As a result, through the cascade use of above three technologies, the proposed biosensing strategy realized sensitive detection of target DNA with a low detection limit of 0.35 fM (S/N = 3) and wide linear range (0.5 fM-500 pM). SIGNIFICANCE: The proposed novel dTMSD combining multiple signal amplification strategies for electrochemical detection of CYFRA21-1 DNA with easy operation not only possesses excellent sensitivity and selectivity, but also has potential application value for monitoring DNA in serum. Meanwhile, the development of highly sensitive and specific CYFRA21-1 DNA detection methods is very important for the prevention and treatment of lung cancer.

Recent advances of NFATc1 in rheumatoid arthritis-related bone destruction: mechanisms and potential therapeutic targets.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease characterized by inflammation of the synovial tissue and joint bone destruction, often leading to significant disability. The main pathological manifestation of joint deformity in RA patients is bone destruction, which occurs due to the differentiation and proliferation of osteoclasts. The transcription factor nuclear factor-activated T cell 1 (NFATc1) plays a crucial role in this process. The regulation of NFATc1 in osteoclast differentiation is influenced by three main factors. Firstly, NFATc1 is activated through the upstream nuclear factor kappa-B ligand (RANKL)/RANK signaling pathway. Secondly, the Ca2+-related co-stimulatory signaling pathway amplifies NFATc1 activity. Finally, negative regulation of NFATc1 occurs through the action of cytokines such as B-cell Lymphoma 6 (Bcl-6), interferon regulatory factor 8 (IRF8), MAF basic leucine zipper transcription factor B (MafB), and LIM homeobox 2 (Lhx2). These three phases collectively govern NFATc1 transcription and subsequently affect the expression of downstream target genes including TRAF6 and NF-κB. Ultimately, this intricate regulatory network mediates osteoclast differentiation, fusion, and the degradation of both organic and inorganic components of the bone matrix. This review provides a comprehensive summary of recent advances in understanding the mechanism of NFATc1 in the context of RA-related bone destruction and discusses potential therapeutic agents that target NFATc1, with the aim of offering valuable insights for future research in the field of RA. To assess their potential as therapeutic agents for RA, we conducted a drug-like analysis of potential drugs with precise structures.

The Progress of Poststroke Seizures.

A stroke is one of the most common fatal diseases of the nervous system, and the number of strokes per year has increased substantially in recent years. Epilepsy is a poststroke complication that greatly affects the prognosis of patients and reduces their quality of survival. Effective avoidance of causative factors can reduce the risk of a poststroke seizure. However, while many studies have been devoted to elucidating the pathogenesis of poststroke seizures, the literature lacks a comprehensive understanding of the pathogenic mechanism. This article briefly presents the current definition, risk factors, pathogenesis, and prognosis of poststroke seizures based on reported studies and literature reviews, aiming to enrich the available knowledge of this disease.

Tuberous sclerosis complex: a case report and literature review.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder with different initial symptoms and complex clinical manifestations. A 14-year-old female patient presented with persistent fever and severe headache. Medical imaging examinations revealed multiple abnormal intracranial lesions. The patient had previously been misdiagnosed with "encephalitis and acute disseminated encephalomyelitis" after visiting numerous hospitals. Eventually, by combing the characteristics of the case and genetic testing results, the patient was diagnosed with TSC accompanied by Mycoplasma pneumoniae infection. The purpose of this case report and literature review is to improve understanding of the clinical diagnosis and treatment of TSC so as to avoid misdiagnosis, missed diagnosis, and overtreatment.

Effectiveness of ultraviolet-C disinfection systems for reduction of multi-drug resistant organism infections in healthcare settings: A systematic review and meta-analysis.

This study aimed to summarise the findings of the studies assessing the effectiveness of ultraviolet C (UV-C) room disinfection in reducing the incidence rate of healthcare-associated multi-drug-resistant organism (MDRO) infections. A systematic screening was conducted using PubMed, EMBASE, and Scopus for randomised controlled trials (RCTs), quasi-experimental studies, and before-after studies, which assessed the efficacy of the UV-C disinfectant system in reducing the incidence of MDRO infections. A random-effects model was used for the analysis. Effect sizes were described as incidence rate ratio (IRR) with 95% confidence intervals (CI). Nine studies were included, all of which were conducted in the USA. No statistically significant reduction in Clostridioides difficile (CD) (IRR: 0.90, 95% CI; 0.62-1.32) and vancomycin-resistant enterococcal (VRE) infection rates (IRR 0.72, 95% CI; 0.38-1.37) was observed with the use of UV-C, but the risk of Gram-negative rod infection was reduced (IRR 0.82, 95% CI; 0.68-0.99).

Advances in the mTOR signaling pathway and its inhibitor rapamycin in epilepsy.

INTRODUCTION: Epilepsy is one of the most common and serious brain syndromes and has adverse consequences on a patient's neurobiological, cognitive, psychological, and social wellbeing, thereby threatening their quality of life. Some patients with epilepsy experience poor treatment effects due to the unclear pathophysiological mechanisms of the syndrome. Dysregulation of the mammalian target of the rapamycin (mTOR) pathway is thought to play an important role in the onset and progression of some epilepsies. METHODS: This review summarizes the role of the mTOR signaling pathway in the pathogenesis of epilepsy and the prospects for the use of mTOR inhibitors. RESULTS: The mTOR pathway functions as a vital mediator in epilepsy development through diverse mechanisms, indicating that the it has great potential as an effective target for epilepsy therapy. The excessive activation of mTOR signaling pathway leads to structural changes in neurons, inhibits autophagy, exacerbates neuron damage, affects mossy fiber sprouting, enhances neuronal excitability, increases neuroinflammation, and is closely associated with tau upregulation in epilepsy. A growing number of studies have demonstrated that mTOR inhibitors exhibit significant antiepileptic effects in both clinical applications and animal models. Specifically, rapamycin, a specific inhibitor of TOR, reduces the intensity and frequency of seizures. Clinical studies in patients with tuberous sclerosis complex have shown that rapamycin has the function of reducing seizures and improving this disease. Everolimus, a chemically modified derivative of rapamycin, has been approved as an added treatment to other antiepileptic medicines. Further explorations are needed to evaluate the therapeutic efficacy and application value of mTOR inhibitors in epilepsy. CONCLUSIONS: Targeting the mTOR signaling pathway provides a promising prospect for the treatment of epilepsy.

A case report of acute intermittent porphyria leading to severe disability.

Acute intermittent porphyria (AIP) is a rare inherited metabolic disorder resulting from increased production of porphyrins and their precursors, δ-aminolevulinic acid (ALA) and porphobilinogen (PBG), due to deficiencies in the enzymatic activity of the heme synthesis pathway. The disease is typically characterized by a triad of abdominal pain, neurologic impairment symptoms, and psychiatric abnormalities. However, only a small percentage of patients present with this classic triad of symptoms. Our female patient, aged 23, was admitted to the hospital with a 4-year history of abnormal mood episodes and weakness in the limbs for over 1 week. She had a previous medical history of intestinal obstruction. After admission, a cranial MRI revealed reversible posterior leukoencephalopathy imaging manifestations, and the patient exhibited weakness of the extremities, respiratory failure, seizures, and severely reduced serum sodium concentration. The diagnosis of AIP was ultimately confirmed by a positive urine PBG-sunlight test and analysis of HMBS gene variants. The absence of typical triadic signs in acute attacks of AIP can make early recognition of the disease challenging. We present a case with multiple typical clinical manifestations of AIP in the hope of aiding clinicians in fully recognizing acute intermittent porphyria.

An ingenious electrochemical aptasensor for detection of CYFRA 21-1 based on dual-output toehold mediated strand displacement reaction.

Herein, a "signal-on" electrochemical aptasensor based on dual-output toehold mediated strand displacement reaction (TMSDR) was developed to detect cytokeratin fragment antigen 21-1 (CYFRA 21-1). First, the methylene blue (MB) labeled signal probe (SP) was combined with a long template strand (TS) to form the three-strands duplex, which was immobilized on magnetic beads. The sequence of TS was innovatively designed with two toehold zones. Thus, with the presence of target DNA and Fuel strand (F), double SP and target were released through TMSDR. Besides, the released target could participate in the next cycle, achieving the further amplification. The released SP was selected by magnetic separation, then added to the gold electrode (GE) to hybridize with the hairpin DNA. Ultimately, the target can be detected by recording the current response of the MB. Under optimized conditions, the response peak current was linear with the concentration of CYFRA 21-1 in range from 10 pM to 1 µM with detection limit of 8.079 pM. The proposed signal amplification strategy was enzyme-free in homogenous solution and the using of magnetic beads improved the recognition efficiency and simplified the experimental steps, which endowed the sensor with super-sensitivity.

Long Noncoding RNA GUSBP11 Knockdown Alleviates Nasopharyngeal Carcinoma via Regulating miR-1226-3p/TM9SF4 Axis.

Purpose: Long noncoding RNAs (lncRNAs) have been confirmed related to the occurrence and progress of multiple cancers, including cervical cancer nasopharyngeal carcinoma (NPC). This study focused on assessing GUSBP11 effects on NPC progression and exploring possible mechanisms. Materials and Methods: RT-qPCR was conducted for assessing GUSBP11 levels within NPC tissues and cells. CCK-8, colony formation, and Transwell were adopted for examining GUSBP11 impacts on NPC cell proliferation and cell metastasis. RT-qPCR analysis and dual-luciferase reporter assay were conducted for judging the expression interrelation of GUSBP11 and its potential target miR-1226-3p. The same methods were carried out for verifying the inhibiting influences of miR-1226-3p upregulation and its potential target TM9SF4. Results: GUSBP11 levels were upregulated within NPC tissues and cells. GUSBP11 downregulation repressed NPC cell proliferation and cell metastasis. In addition, GUSBP11 targeted and negatively regulated miR-1226-3p. Furthermore, miR-1226-3p targeted TM9SF4 and mediated GUSBP11's impacts on TM9SF4 levels. At last, the authors proved the critical role of the GUSBP11/miR-1226-3p/TM9SF4 axis in regulating NPC progression. Conclusion: These findings indicate that downregulation of GUSBP11 alleviates NPC development by regulating the miR-1226-3p/TM9SF4 axis.

A cilia-independent function of BBSome mediated by DLK-MAPK signaling in C. elegans photosensation.

Bardet-Biedl syndrome (BBS) is a genetic disorder that affects primary cilia. BBSome, a protein complex composed of eight BBS proteins, regulates the structure and function of cilia, and its malfunction causes BBS in humans. Here, we report a cilia-independent function of BBSome. To identify genes that regulate the C. elegans photoreceptor protein LITE-1 in ciliated ASH photosensory neurons, we performed a genetic screen and isolated bbs mutants. Functional analysis revealed that BBSome regulates LITE-1 protein stability independently of cilia. Through another round of genetic screening, we found that this cilia-independent function of BBSome is mediated by DLK-MAPK signaling, which acts downstream of BBSome to control LITE-1 stability via Rab5-mediated endocytosis. BBSome exerts its function by regulating the expression of DLK. BBSome also regulates the expression of LZK, a mammalian DLK in human cells. These studies identify a cilia-independent function of BBSome and uncover DLK as an evolutionarily conserved BBSome effector.

An ultrasensitive label-free photoelectrochemical aptasensor based on terminal deoxynucleotidyl transferase amplification and catalytic reaction of G-quadruplex/hemin.

An ultrasensitive photoelectrochemical (PEC) analysis method based on terminal deoxynucleotidyl transferase (TdT) amplification effect and G-quadruplex/hemin (G4/hemin) catalytic reaction assisted signal amplification was prepared for the determination of ampicillin. A novel Au NPs@SnIn4S8-graphene sensitized structure was used as photoactive material to attain an intense basic photocurrent. In the presence of the target, P1 obtained by strand displacement reaction was introduced into the electrode surface, and abundant G4/hemin was formed in the presence of TdT and hemin. Subsequently, G4/hemin with horseradish peroxidase-mimicking activity catalyzed the oxidation of 4-chloro-1-naphthol by H2O2 to form benzo-4-chlorohexadienone precipitation on the modified electrode surface, which severely hindered the electron transfer and effectively inhibited the photocurrent output. The detection range of the PEC aptasensor for ampicillin was 10 fM-30 nM, and the limit of detection was 4.97 fM. The aptasensor had good stability and high sensitivity, and possessed a promising application in biological analysis and environmental monitoring.

Susac syndrome with the typical clinical triad: A case report and literature review.

Susac syndrome is an immune-mediated microvascular disease characterized by the clinical triad of acute multiple encephalopathies, branch retinal artery occlusion, and sensorineural hearing loss. However, the typical clinical triad is not seen in all patients at disease onset. In this study, a 29-year-old male was admitted to our hospital due to aggravation of headache accompanied by retarded reaction. After treatment for a diagnosis of possible central nervous system vasculitis, the patient's retarded reaction and neurological dysfunction were improved. One year after discharge, the patient had no abnormal clinical symptoms and he discontinued taking prednisone voluntarily five months after discharge. Two years later, the patient was admitted to our hospital again owing to a sudden visual field defect in the superonasal quadrant of the left eye for one week, and Susac syndrome was diagnosed. After treatment, the patient's condition became stabilized with no further progress, but the visual field defect did not recover. At the onset of Susac syndrome, the typical clinical triad of Susac syndrome is rare, so this disease is difficult to be recognized at the beginning. The case we report presented the clinical triad two years after the disease onset. We expect that this case report will increase physicians' understanding of Susac syndrome.

Delayed multiple intracranial aneurysms caused by left atrial myxoma: a case report and literature review.

Intracranial aneurysm may appear even after the removal of the cardiac myxoma. However, the pathogenesis and treatment of such aneurysm lesions are not clear. The study aimed to explore the clinical and imaging manifestation, hypothetical pathogenesis, and therapy in one case of left atrial myxoma causing multiple intracranial aneurysms. A 14-year-old male displayed a 3-hour history of episodic loss of consciousness and right hemiplegia after a leapfrog-like movement. The myxoma was diagnosed by a combination of clinical examination, leading to the diagnosis of mitral dynamic obstruction with a Grade III mitral diastolic murmur and tumor plop; magnetic resonance imaging, revealing multiple ischemic sites in both semi-oval centers; and transthoracic echocardiography, demonstrating a mitral valve obstruction. The myxoma was removed surgically; however, computed tomography angiography showed multiple intracranial aneurysms in both middle cerebral arteries 18 months after resection of the atrial myxoma. After conservative treatment, the patient had no neurological dysfunction symptoms for 5 years after myxoma resection. His condition is relatively stable. In conclusion, resection of the atrial myxoma may eliminate the early neurological symptoms, but it cannot ensure the nonoccurrence of delayed intracranial aneurysms. The neoplastic process theory was favored for explaining the aneurysm development in this case. According to the specific conditions of the patient, a combination of open surgery, chemotherapy, radiotherapy, and coil embolization is recommended.

Regulated bioanalysis of liposomal amphotericin B to support pharmacokinetic studies of liposomal drugs.

Background: Because of the delicate nature of liposomes, bioanalysis of free and liposomal-encapsulated drugs is among the most challenging assays to perform. Current regulatory guidance for bioanalysis is not sufficient to address the complexity of this particular formulation. Method & results: Three individual LC-MS/MS methods to quantify free amphotericin B (10-3000 ng/ml) and encapsulated amphotericin B (100-50,000 ng/ml) in pretreated human plasma and total amphotericin B (100-50,000 ng/ml) in human plasma were fully validated and applied to a bioequivalence study. The acceptance criteria and experimental design of additional validation tests using cross quality control were carefully deliberated a priori and included in the sample analysis as well. Discussion: Additional validation tests are necessary to demonstrate that the measured concentration of the intended component is accurate and free of interference from other coexisting components in the sample. These practices can be used as guidance for future liposomal drug method validation.

Lithiophilic Carbon Nanofiber/Graphene Nanosheet Composite Scaffold Prepared by a Scalable and Controllable Biofabrication Method for Ultrastable Dendrite-Free Lithium-Metal Anodes.

Li metal is regarded as a promising anode for high-energy-density Li batteries, while the limited cycle life and fast capacity decay caused by notorious Li dendrite growth seriously impedes its application. Herein, a robust and highly lithiophilic bacterial cellulose-derived carbon nanofiber@reduced graphene oxide nanosheet (BC-CNF@rGO) composite scaffold is fabricated as a host for dendrite-free Li metal anode through an in situ biofabrication method. The abundant lithiophilic functional groups, conductive 3D network, and excellent mechanical property can effectively regulate uniform Li nucleation and deposition, enable fast reaction kinetics, and alleviate volume change. As a result, the BC-CNF@rGO skeleton achieves exceptional Li plating/stripping performance with a high average Coulombic efficiency of 98.3% over 800 cycles, and a long cycle life span of 5000 h at 2 mA cm-2 @1 mAh cm-2 with a low overpotential of ≈15 mV for lithium plating. Furthermore, full cells coupling BC-CNF@rGO-Li anode with LiFePO4 cathode achieves an unprecedented cycling stability with a long cycle life of 3000 cycles at 1 C. This work sheds light on a promising material design and fabrication strategy for realizing high performance Li metal batteries.

Ultrathin, Strong, and Highly Flexible Ti3C2Tx MXene/Bacterial Cellulose Composite Films for High-Performance Electromagnetic Interference Shielding.

The fabrication of ultrathin films that are electrically conductive and mechanically strong for electromagnetic interference (EMI) shielding applications is challenging. Herein, ultrathin, strong, and highly flexible Ti3C2Tx MXene/bacterial cellulose (BC) composite films are fabricated by a scalable in situ biosynthesis method. The Ti3C2Tx MXene nanosheets are uniformly dispersed in the three-dimensional BC network to form a mechanically entangled structure that endows the MXene/BC composite films with excellent mechanical properties (tensile strength of 297.5 MPa at 25.7 wt % Ti3C2Tx) and flexibility. Importantly, a 4 µm thick Ti3C2Tx/BC composite film with 76.9 wt % Ti3C2Tx content demonstrates a specific EMI shielding efficiency of 29141 dB cm2 g-1, which surpasses those of most previously reported MXene-based polymer composites with similar MXene contents and carbon-based polymer composites. Our findings show that the facile, environmentally friendly, and scalable fabrication method is a promising strategy for producing ultrathin, strong, and highly flexible EMI shielding materials such as the freestanding Ti3C2Tx/BC composite films for efficient EMI shielding to address EMI problems of a fast-developing modern society.

Constrains for seeking post-abortion care among adolescents and young women in Guangzhou, China: a cross-sectional study.

BACKGROUND: In China, post-abortion care (PAC) services mainly focus on married couples, such that adolescents and unmarried young womenhave limited access to those services for contraception counseling. The provision of youth-friendly PAC services in public hospitals is a new concept in China. This study examined the magnitude of PAC services utilization as well as factors influencing it's uptake among adolescents and young women in Guangzhou, China. METHODS: A cross-sectional study was performed from 1st March 2020 to 30th September 2020 using anonymous self-administered questionnaire among 688 women aged 15-24 years in Tianhe district, Guangzhou. The Multivariate logistic regression was used to determine factors that were significantly associated with the uptake of PAC services. RESULTS: The magnitude of PAC services utilization was 35.9% among adolescents and young women in Guangzhou, China. Students were 69.0% significantly less likely to use PAC services compared to women who had no job. Immigrants were 59.0% significantly less likely to use PAC services than their native counterparts. Women who had a feeling of stigma were 70.0% significantly less likely to use PAC services compared to those who did not feel stigmatized. CONCLUSIONS: The study highlights the need to strengthen youth-friendly PAC services provision, and emphasizes the importance of education about both family planning and abortion services among disadvantaged sub-groups of women in the study setting.

Clinical and Imaging Prognosis in Patients with Delayed Encephalopathy after Acute Carbon Monoxide Poisoning.

OBJECTIVE: To study the clinical manifestations, magnetic resonance imaging (MRI) findings, and prognosis of delayed encephalopathy after carbon monoxide poisoning (DEACMP). METHODS: The medical records of 20 patients with DEACMP were retrospectively reviewed. All the patients received hyperbaric oxygen treatment and other treatments as necessary. RESULTS: The patients had diverse clinical manifestations, including memory deficits, personality changes, cognitive or executive function deficits, mood disorders, Parkinsonism, dystonia or other motor impairments, and akinetic mutism. MRI revealed lesions in the bilateral cerebral white matter and/or basal ganglia. Except for the pathologically confirmed DEACMP, epileptic seizure, hemiplegia, and vegetative state, the remaining symptoms had been improved, especially the cognitive impairment, which had been decreased from 95% to 25% and psychiatric symptoms also decreased from 95% to 55% at the 6-month follow-up. CONCLUSIONS: The prognosis of patients with DEACMP was poor, and they had a relatively severe disability. The early use of hyperbaric oxygen is of great significance to improve clinical efficacy and get a better prognosis.

Direct quantitation of free, encapsulated, total doxorubicin and doxorubicinol in stabilized frozen human plasma to support a BE study of liposomal doxorubicin.

Regulatory guidance requires the quantification of encapsulated and free doxorubicin for a liposomal doxorubicin injection bioequivalence study. Due to the instability of liposome formulations in plasma samples, the release of free drug from the liposomal encapsulated doxorubicin during sample handling would result in elevation of measured free doxorubicin concentration. To prevent the potential release of free drug, stabilizer reagents and procedures were successfully developed and validated to adequately stabilize liposomal drugs in plasma samples during sample collection, storage and extraction. Three LC-MS/MS methods were developed and fully validated for direct quantitation of free, encapsulated and total doxorubicin concentrations in human plasma according to relevant regulatory guidance: Method 1: Quantitation of free doxorubicin and doxorubicinol at a linear range of 1-400 ng/mL and 0.5-10 ng/mL, respectively, from stabilizer treated plasma samples using solid phase extraction (SPE); Method 2: Quantitation of encapsulated doxorubicin at a linear range of 50-50,000 ng/mL from the stabilizer treated plasma sample using SPE followed by PPE extraction method; Method 3: Quantitation of total concentration of doxorubicin from untreated plasma samples at a linear range of 50-50,000 ng/mL using PPE. All three methods were successfully used to support a bioequivalence study between Caelyx® and Duomeisu® (Doxorubicin Hydrochloride Liposomal injection, generic doxorubicin formulation produced by CSPC). Incurred sample reanalysis (ISR) passing rate for total doxorubicin, free doxorubicin/doxorubicinol, and encapsulated doxorubicin methods were 100 %, 84.7 %/100 %, and 98.5 %, respectively. The measured total doxorubicin concentrations matched the sum of free and encapsulated doxorubicin concentrations.