Electroacupuncture Reduces Fibromyalgia Pain via Neuronal/Microglial Inactivation and Toll-like Receptor 4 in the Mouse Brain: Precise Interpretation of Chemogenetics.

Fibromyalgia (FM) is a complex, chronic, widespread pain syndrome that can cause significant health and economic burden. Emerging evidence has shown that neuroinflammation is an underlying pathological mechanism in FM. Toll-like receptors (TLRs) are key mediators of the immune system. TLR4 is expressed primarily in microglia and regulates downstream signaling pathways, such as MyD88/NF-κB and TRIF/IRF3. It remains unknown whether electroacupuncture (EA) has therapeutic benefit in attenuating FM pain and what role the TLR4 pathway may play in this effect. We compared EA with sham EA to eliminate the placebo effect due to acupuncture. We demonstrated that intermittent cold stress significantly induced an increase in mechanical and thermal FM pain in mice (mechanical: 2.48 ± 0.53 g; thermal: 5.64 ± 0.32 s). EA but not sham EA has an analgesic effect on FM mice. TLR4 and inflammatory mediator-related molecules were increased in the thalamus, medial prefrontal cortex, somatosensory cortex (SSC), and amygdala of FM mice, indicating neuroinflammation and microglial activation. These molecules were reduced by EA but not sham EA. Furthermore, a new chemogenetics method was used to precisely inhibit SSC activity that displayed an anti-nociceptive effect through the TLR4 pathway. Our results imply that the analgesic effect of EA is associated with TLR4 downregulation. We provide novel evidence that EA modulates the TLR4 signaling pathway, revealing potential therapeutic targets for FM pain.

Psychological Outcomes and Quality of Life of Fibromyalgia Patients with Vitamin D Supplementation-A Meta-Analysis.

The efficacy of current pharmaceutical treatments for fibromyalgia are limited. Vitamin D has shown promise in relieving pain. However, there is a lack of comprehensive analysis of psychological outcomes with vitamin D supplementation in fibromyalgia. This study aimed to investigate the impact of vitamin D supplementation on psychological outcomes and quality of life in fibromyalgia patients, given the unmet clinical need for effective treatment options. A meta-analysis of randomized controlled trials comparing vitamin D to placebo and prospective studies examining changes before and after vitamin D supplementation for patients with fibromyalgia was conducted to evaluate the effects of vitamin D on psychological outcomes, quality of life, and pain scores in patients with fibromyalgia. Databases were searched for relevant articles published from earliest available date to October 31, 2022. (PROSPERO number, CRD42022369889). We included 8 trials with a total of 694 participants and found that vitamin D supplementation had significant positive effects on physical function (standard mean differences (SMD) = 0.44, 95% CI = [0.10, 0.77 ]), role limitations due to emotional health (SMD = 0.57, 95% CI = [0.32, 0.82]), social function (SMD = 0.50, 95% CI = [0.08, 0.93]), and general health (SMD = 0.36, 95% CI = [0.11, 0.61]). Improvement of the Fibromyalgia Impact Questionnaire (FIQ) scores was noted (SMD = -0.414, 95% CI = [-0.808, -0.021]), but not on the Visual Analog Scale (VAS) (SMD = -0.15, 95% CI = [-0.771, 0.471]) and the Beck's Depression Inventory (BDI) scores (SMD = -0.456, 95% CI = [-1.27, 0.30]). In conclusion, vitamin D supplementation might be an alternative option for improvement of psychological outcomes and quality of life in patients with fibromyalgia.

Anti-Inflammatory Effect of Traditional Chinese Medicine on the Concept of Mind-Body Interface.

In this chapter, we conducted a systemic literature review for the anti-inflammatory effects of Traditional Chinese Medicine (TCM) applying molecular mechanisms focusing on the neuroinflammation and gut-brain axis in three neuropsychiatric disorders: major depressive disorder, Alzheimer's disease, and Parkinson's disease. We demonstrated the anti-inflammation or immunomodulation effects of TCM, including acupuncture, from basic and clinical research, including cellular and molecular approaches. In conclusion, inflammation plays a critical role in the neuropsychopathological process. At the same time, anti-inflammation seems to be the common biological pathway for the effects of TCM and acupuncture in depression, Alzheimer's disease, and Parkinson's disease.

Rapid progressive vaccine-induced immune thrombotic thrombocytopenia with cerebral venous thrombosis after ChAdOx1 nCoV-19 (AZD1222) vaccination: A case report.

BACKGROUND: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare and potentially life-threatening condition after receiving coronavirus disease vaccines. It is characterized by symptom onset at 5 to 30 d postvaccination, thrombocytopenia, thrombosis, high D-dimer level, and antiplatelet factor 4 (anti-PF4) antibody positivity. VITT can progress very fast, requiring urgent management. Only few studies have described its detailed clinical course and imaging changes. We report a typical VITT case in a patient who underwent regular repeated brain imaging examinations. CASE SUMMARY: A young woman presented with headaches at 7 d after the ChAdOx1 nCoV-19 vaccine (AZD1222) injection. She then showed progressive symptoms of left upper limb clumsiness. Brain computed tomography revealed venous infarction at the right parietal lobe with a hyperacute thrombus in the cortical vein. Two hours later, brain magnetic resonance imaging revealed hemorrhage at the same area. Magnetic resonance venography showed an irregular contour of the right transverse sinus. Laboratory examination revealed a high D-dimer level, thrombocytopenia, and a high titer for anti-PF4 antibodies. She was treated with anticoagulants, intravenous immunoglobulin, and steroids and analgesic agents were administered for pain control. She had a marked improvement on headaches and clumsiness after treatment along with radiological thrombus resolution. During follow-up at the outpatient department, her modified Rankin scale at 90 d was 1. CONCLUSION: Clinicians should be alerted whenever patients present with persistent and progressive headaches or focal motor/sensory deficits postvaccination.

Predictors of unfavorable outcome at 90 days in basilar artery occlusion patients.

BACKGROUND: In a previous study, basilar artery occlusion (BAO) was shown to lead to death or disability in 80% of the patients. The treatment for BAO patients in the acute stage includes thrombolysis and intra-arterial thrombectomy, but not all patients benefit from these treatments. Thus, understanding the predictors of outcome before initiating these treatments is of special interest. AIM: To determine the predictors related to the 90-d clinical outcome in patients with BAO in an Asian population. METHODS: We performed a retrospective case review of patients admitted to a tertiary stroke center between 2015 and 2019. We used the international classification of diseases-10 criteria to identify cases of posterior circulation stroke. A neurologist reviewed every case, and patients fulfilling the criteria defined in the Basilar Artery International Cooperation Study were included. We then analyzed the patients' characteristics and factors related to the 90-d outcome. RESULTS: We identified a total of 99 patients as real BAO cases. Of these patients, 33 (33.3%) had a favorable outcome at 90 d (modified Rankin Scale: 0-3). Moreover, 72 patients received intra-arterial thrombectomy, while 13 patients received intravenous tissue-type plasminogen activator treatment. We observed a favorable outcome in 33.3% of the cases and an unfavorable outcome in 66.7% of the cases. We found that the initial National Institutes of Health Stroke Scale (NIHSS) score and several BAO symptoms, including impaired consciousness, tetraparesis, and pupillary abnormalities, were significantly associated with an unfavorable outcome (P < 0.05), while cerebellar symptoms were associated with a favorable outcome (P < 0.05). In the receiver operating characteristic (ROC) analysis, the areas under the ROC curve of initial NIHSS score, impaired consciousness, tetraparesis, cerebellar symptoms, and pupillary abnormalities were 0.836, 0.644, 0.727, 0.614, and 0.614, respectively. Initial NIHSS score showed a higher AUROC (0.836) compared to BAO symptoms. CONCLUSION: The most important predictor of an unfavorable outcome was the initial NIHSS score. BAO symptoms, including tetraparesis, impaired consciousness, and pupillary abnormality were also related to an unfavorable outcome.

Glutamic acid decarboxylase 65-positive autoimmune encephalitis presenting with gelastic seizure, responsive to steroid: A case report.

BACKGROUND: Anti-glutamic acid decarboxylase (GAD) antibody is known to cause several autoimmune-related situations. The most known relationship is that it may cause type I diabetes. In addition, it was also reported to result in several neurologic syndromes including stiff person syndrome, cerebellar ataxia, and autoimmune encephalitis. Decades ago, isolated epilepsy associated with anti-GAD antibody was first reported. Recently, the association between temporal lobe epilepsy and anti-GAD antibody has been discussed. Currently, with improvements in examination technique, many more autoimmune-related disorders can be diagnosed and treated easier than in the past. CASE SUMMARY: A 44-year-old female Asian with a history of end-stage renal disease (without diabetes mellitus) under hemodialysis presented with diffuse abdominal pain. The initial diagnosis was peritonitis complicated with sepsis and paralytic ileus. Her peritonitis was treated and she recovered well, but seizure attack was noticed during hospitalization. The clinical impression was gelastic seizure with the presentation of frequent smiling, head turned to the right side, and eyes staring without focus; the duration was about 5-10 s. Temporal lobe epilepsy was recorded through electroencephalogram, and she was later diagnosed with anti-GAD65 antibody positive autoimmune encephalitis. Her seizure was treated initially with several anticonvulsants but with poor response. However, she showed excellent response to intravenous methylprednisolone pulse therapy. Her consciousness returned to normal, and no more seizures were recorded after 5 d of intravenous methylprednisolone treatment. CONCLUSION: In any case presenting with new-onset epilepsy, in addition to performing routine brain imaging to exclude structural lesion and cerebrospinal fluid studies to exclude common etiologies of infection and inflammation, checking the autoimmune profile has to be considered. In the practice of modern medicine, autoimmune-related disorders are relatively treatable and should not be missed.

Use of a Smartphone Application to Speed Up Interhospital Transfer of Acute Ischemic Stroke Patients for Thrombectomy.

Background: In most countries, large cerebral artery occlusion is identified as the leading cause of disability. In 2015, five large-scale clinical trials confirmed the benefit of intra-arterial thrombectomy. However, thrombectomy is a highly technical and facility-dependent procedure. Primary stroke centers need to transfer patients to comprehensive stroke centers to perform thrombectomy. The time-lapse during interhospital transfer would decrease the chance of the patient's proper recovery. Communication barriers also contribute to this delay. Aims: We used a smartphone application to overcome communication barriers between hospitals. We aimed to shorten the door-to-puncture time of interhospital transfer patients. Methods: We began using a smartphone application, "LINE," to facilitate interhospital communication on May 01, 2018. We carried out retrospective data analyses for all the transfer patients (n = 351), with the primary outcome being the door-to-puncture time in our comprehensive stroke center (China Medical University Hospital). We compared the three periods: May 01 to Dec 31, 2017 (before the use of the smartphone application); May 01 to Dec 31, 2018 (the 1st year of using the smartphone application); and May 01 to Dec 31, 2019 (the 2nd year of using the smartphone application). We also compared the transfer data with non-transfer thrombectomies in the same period. Results: We compared 2017, 2018, and 2019 data. The total number of transfer patients increased over the years: 63, 113, 175, respectively. The mean door-to-puncture time decreased significantly, going from 109, through 102, to 92 min. Meanwhile, the mean door-to-puncture time in non-transfer patients were 140.3, 122.1, and 129.3 min. The main reason of time saving was the change of the way of communication, from point-to-point interhospital communication to hub-to-spoke interhospital communication. Conclusions: We used this smartphone application to enhance interhospital communication, changed from the point-to-point to hub-to-spoke method. It made us overcome the communication barrier and build up interhospital connection, thus shortening the door-to-puncture time. Our experience demonstrated the importance of close communication and teamwork in hyperacute stroke care, especially in interhospital transfer for thrombectomy.

Three-year mortality in cryptococcal meningitis: Hyperglycemia predict unfavorable outcome.

Existing evidence revealed grave prognosis for cryptococcal meningitis (CM), particularly its short-term mortality. However, its long-term survival and prognostic factors remained unknown. This study investigated 3-year mortality and analyzed its predictive factors in patients with CM. This retrospective cohort study with 83 cerebrospinal fluid culture-confirmed CM patients was conducted at China Medical University Hospital from 2003 to 2016. The 3-year mortality rate in patients with CM was 54% (45 deaths among 83 patients). Advanced age, human immunodeficiency virus (HIV) seronegative state, low Glasgow Coma Scale score on admission, decreased hemoglobin and hyperglycemia on diagnosis were associated with 3-year mortality. After multivariate adjustment in the Cox proportional hazard model, only severe hyperglycemia (serum glucose ≥200 mg/dL) on diagnosis could predict 3-year mortality.

Transient receptor potential V1 modulates neuroinflammation in Parkinson's disease dementia: Molecular implications for electroacupuncture and rivastigmine.

OBJECTIVES: Parkinson's disease (PD) is a common progressive neurodegeneration disease. Its incidence increases with age and affects about 1% of people over 60. Incidentally, transient receptor potential V1 (TRPV1) and its relation with neuroinflammation in mouse brain has been widely reported. MATERIALS AND METHODS: We used 6-hydroxydopamine (6-OHDA) to induce PDD in mice. We then used the Morris water maze and Bio-Plex to test learning and inflammatory mediators in mouse plasma. Western blotting and immunostaining were used to examine TRPV1 pathway in the hippocampus and medial prefrontal cortex (mPFC). RESULTS: On acquisition days 3 (Control = 4.40 ± 0.8 sec, PDD = 9.82 ± 1.52 sec, EA = 5.04 ± 0.58 sec, Riva = 4.75 ± 0.87 sec; P=0.001) and 4, reversal learning days 1, 2, 3 (Control = 2.86 ± 0.46 sec, PDD = 9.80 ± 1.83 sec, EA = 4.6 ± 0.82 sec, Riva = 4.6 ± 1.03 sec; P=0.001) and 4, PDD mice showed significantly longer escape latency than the other three groups. Results showed that several cytokines were up-regulated in PDD mice and reversed by EA and rivastigmine. TRPV1 and downstream molecules were up-regulated in PDD mice and further reversed by EA and rivastigmine. Interestingly, α7 nicotinic receptors and parvalbumin levels in both the hippocampus and prefrontal cortex increased in EA-treated mice, but not in rivastigmine-treated mice. CONCLUSION: Our results showed that TRPV1 played a role in the modulation of neuroinflammation of PDD, and could potentially be a new target for treatment.

Acupuncture reduced the medical expenditure in migraine patients: Real-world data of a 10-year national cohort study.

OBJECTIVES: According to the data of Organisation for Economic Cooperation and Development, almost all the countries got increased medical expenditures in these years. Among the diseases, migraine is a condition that affects predominantly young and middle-aged people. It results in great economic losses. So we perform this research to investigate the acupuncture effect of reducing medical expenditure and medical resources use. PERSPECTIVE: Acupuncture is a non-pharmacologic treatment and it became popular in recent years. In Taiwan, about 13% migraine patients visited acupuncture doctor. We hypothesized that the acupuncture had the additional effect than the medical treatment. SETTING: We analysed the economic cost and medical visits in the real word. METHODS: We used national cohort data from Taiwan, retrospectively gathered between 2000 and 2010. We selected newly diagnosed migraine patients who were diagnosed by registered neurologists formally licensed by the Taiwan Neurological Society. We divided these patients into two groups: with and without acupuncture treatment. The main outcome was medical expenditures and visits within 1 year after acupuncture. RESULTS: In migraine patients who received acupuncture treatment, medical expenditures on emergency care and hospitalization were significantly lower than the group without acupuncture treatment. CONCLUSION: According to our real-world data, acupuncture can reduce the medical expenditure in migraine patients within 1 year after diagnosis. For the health policy maker, it is cost effective to encourage combining acupuncture and western medicine to treat migraine patients. For the doctors in routine clinical practice, who may consider to consult acupuncture doctors to deal with the migraine patients together.

The Neurologic Manifestations of Coronavirus Disease 2019 Pandemic: A Systemic Review.

Objective: Review and integrate the neurologic manifestations of the Coronavirus Disease 2019 (COVID-19) pandemic, to aid medical practitioners who are combating the newly derived infectious disease. Methods: We reviewed the clinical research, consisting of mainly case series, on reported neurologic manifestations of COVID-19. We also reviewed basic studies to understand the mechanism of these neurologic symptoms and signs. Results: We included 79 studies for qualitative synthesis and 63 studies for meta-analysis. The reported neurologic manifestations were olfactory/taste disorders (35.6%), myalgia (18.5%), headache (10.7%), acute cerebral vascular disease (8.1%), dizziness (7.9%), altered mental status (7.8%), seizure (1.5%), encephalitis, neuralgia, ataxia, Guillain-Barre syndrome, Miller Fisher syndrome, intracerebral hemorrhage, polyneuritis cranialis, and dystonic posture. Conclusions: Neurologic manifestations in COVID-19 may alert physicians and medical practitioners to rule in high-risk patients. The increasing incidence of olfactory/taste disorders, myalgia, headache, and acute cerebral vascular disease renders a possibility that COVID-19 could attack the nervous system. The cytokine secretion and bloodstream circulation (viremia) are among the most possible routes into the nervous system.

EFHD2 contributes to non-small cell lung cancer cisplatin resistance by the activation of NOX4-ROS-ABCC1 axis.

Recurrence and metastasis remain the major cause of cancer mortality. Even for early-stage lung cancer, adjuvant chemotherapy yields merely slight increase to patient survival. EF-hand domain-containing protein D2 (EFHD2) has recently been implicated in recurrence of patients with stage I lung adenocarcinoma. In this study, we investigated the correlation between EFHD2 and chemoresistance in non-small cell lung cancer (NSCLC). High expression of EFHD2 was significantly associated with poor overall survival of NSCLC patients with chemotherapy in in silica analysis. Ectopic EFHD2 overexpression increased cisplatin resistance, whereas EFHD2 knockdown improved chemoresponse. Mechanistically, EFHD2 induced the production of NADPH oxidase 4 (NOX4) and in turn the increase of intracellular reactive oxygen species (ROS), consequently activating membrane expression of the ATP-binding cassette subfamily C member 1 (ABCC1) for drug efflux. Non-steroidal anti-inflammatory drug (NSAID) ibuprofen suppressed EFHD2 expression by leading to the proteasomal and lysosomal degradation of EFHD2 through a cyclooxygenase (COX)-independent mechanism. Combining ibuprofen with cisplatin enhanced antitumor responsiveness in a murine xenograft model in comparison with the individual treatment. In conclusion, we demonstrate that EFHD2 promotes chemoresistance through the NOX4-ROS-ABCC1 axis and therefore developing EFHD2-targeting strategies may offer a new avenue to improve adjuvant chemotherapy of lung cancer.

Propofol pump controls nonconvulsive status epilepticus in a hepatic encephalopathy patient: A case report.

BACKGROUND: Status epilepticus is an emergent and critical condition which needs management without hesitation. Nonconvulsive status epilepticus (NCSE) tends to be less recognized, and its diagnosis is delayed in comparison with overt status epilepticus because of the absence of specific clinical signs. It is often difficult to make a diagnosis, particularly in patients with hepatic encephalopathy. CASE SUMMARY: A 38-year-old man with a history of alcoholic liver cirrhosis presented with altered mental status; the initial diagnosis was hepatic encephalopathy. Although optimal treatment for hepatic encephalopathy was administered, the patient's mental status did not improve. A final diagnosis of NCSE was made by continuous electroencephalogram (EEG) monitoring. Treatment with levetiracetam and propofol pump was immediately started. The patient's consciousness gradually improved after discontinuation of propofol therapy, and no further epileptic discharge was observed by EEG monitoring. After 1 wk, the patient returned to full consciousness, and he was able to walk in the hospital ward without assistance. He was discharged with minimal sequela of bilateral conjunctivitis. CONCLUSION: In cases of persistent altered mental status without reasonable diagnosis, NCSE should be considered in hepatic encephalopathy patients with persistently altered levels of consciousness, and EEG monitoring is very important. We also recommend propofol as a safe and efficient therapy for NCSE in liver cirrhosis patients.

Immunoglobulin G4-Related Disease Presented as Recurrent Otitis Media and Mixed Hearing Loss Treated With Cyclophosphamide and Rituximab: A Case Report.

Immunoglobulin G4-related disease (IgG4-RD) is a systemic autoimmune disease; however, it rarely presents as recurrent otitis media and mixed hearing loss. In this article, we present a 43-year-old male patient who presented with recurrent otitis media and mixed hearing loss that is the 12th case of IgG4-RD with middle and inner ear involvement. We also report the clinical response of cyclophosphamide and rituximab therapy in IgG4- RD. These two drugs have never been used to treat otologic symptoms, but were used to treat other organs affected by IgG4-RD. One year later, the patient underwent mastoidectomy of the right ear and the pathological reports revealed IgG4-related disease. A rheumatologist administered immunosuppressive therapy comprising cyclophosphamide and rituximab. After therapy, patient's right-sided mixed hearing loss partially improved. We recorded all pure tone audiometry data to evaluate the clinical course and treatment effect. Finally, we concluded that pathological confirmation and further immunosuppressive therapy should be considered in a timely manner to prevent hearing impairment. We recommend cyclophosphamide and rituximab for the treatment of diseases involving the middle and inner ear.

ICAM1 Is a Potential Cancer Stem Cell Marker of Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) accounts for about 90% of esophageal cancer diagnosed in Asian countries, with its incidence on the rise. Cancer stem cell (CSC; also known as tumor-initiating cells, TIC) is inherently resistant to cytotoxic chemotherapy and radiation and associates with poor prognosis and therapy failure. Targeting therapy against cancer stem cell has emerged as a potential therapeutic approach to develop effective regimens. However, the suitable CSC marker of ESCC for identification and targeting is still limited. In this study, we screened the novel CSC membrane protein markers using two distinct stemness characteristics of cancer cell lines by a comparative approach. After the validation of RT-PCR, qPCR and western blot analyses, intercellular adhesion molecule 1 (ICAM1) was identified as a potential CSC marker of ESCC. ICAM1 promotes cancer cell migration, invasion as well as increasing mesenchymal marker expression and attenuating epithelial marker expression. In addition, ICAM1 contributes to CSC properties, including sphere formation, drug resistance, and tumorigenesis in mouse xenotransplantation model. Based on the analysis of ICAM1-regulated proteins, we speculated that ICAM1 regulates CSC properties partly through an ICAM1-PTTG1IP-p53-DNMT1 pathway. Moreover, we observed that ICAM1 and CD44 could have a compensation effect on maintaining the stemness characteristics of ESCC, suggesting that the combination of multi-targeting therapies should be under serious consideration to acquire a more potent therapeutic effect on CSC of ESCC.

Improved silicon nanowire field-effect transistors for fast protein-protein interaction screening.

Understanding how proteins interact with each other is the basis for studying the biological mechanisms behind various physiological activities. Silicon nanowire field-effect transistors (SiNW-FETs) are sensitive sensors used to detect biomolecular interactions in real-time. However, the majority of the applications that use SiNW-FETs are for known interactions between different molecules. To explore the capability of SiNW-FETs as fast screening devices to identify unknown interacting molecules, we applied mass spectrometry (MS) to analyze molecules reversibly bound to the SiNW-FETs. Calmodulin (CaM) is a Ca(2+)-sensing protein that is ubiquitously expressed in cells and its interaction with target molecules is Ca(2+)-dependent. By modifying the SiNW-FET surface with glutathione, glutathione S-transferase (GST)-tagged CaM binds reversibly to the SiNW-FET. We first verified the Ca(2+)-dependent interaction between GST-CaM and purified troponin I, which is involved in muscle contraction, through the conductance changes of the SiNW-FET. Furthermore, the cell lysate containing overexpressed Ca(2+)/CaM-dependent protein kinase IIα induced a conductance change in the GST-CaM-modified SiNW-FET. The bound proteins were eluted and subsequently identified by MS as CaM and kinase. In another example, candidate proteins from neuronal cell lysates interacting with calneuron I (CalnI), a CaM-like protein, were captured with a GST-CalnI-modified SiNW-FET. The proteins that interacted with CalnI were eluted and verified by MS. The Ca(2+)-dependent interaction between GST-CalnI and one of the candidates, heat shock protein 70, was re-confirmed via the SiNW-FET measurement. Our results demonstrate the effectiveness of combining MS with SiNW-FETs to quickly screen interacting molecules from cell lysates.

Label-free quantitative proteomics of CD133-positive liver cancer stem cells.

BACKGROUND: CD133-positive liver cancer stem cells, which are characterized by their resistance to conventional chemotherapy and their tumor initiation ability at limited dilutions, have been recognized as a critical target in liver cancer therapeutics. In the current work, we developed a label-free quantitative method to investigate the proteome of CD133-positive liver cancer stem cells for the purpose of identifying unique biomarkers that can be utilized for targeting liver cancer stem cells. Label-free quantitation was performed in combination with ID-based Elution time Alignment by Linear regression Quantitation (IDEAL-Q) and MaxQuant. RESULTS: Initially, IDEAL-Q analysis revealed that 151 proteins were differentially expressed in the CD133-positive hepatoma cells when compared with CD133-negative cells. We then analyzed these 151 differentially expressed proteins by MaxQuant software and identified 10 significantly up-regulated proteins. The results were further validated by RT-PCR, western blot, flow cytometry or immunofluorescent staining which revealed that prominin-1, annexin A1, annexin A3, transgelin, creatine kinase B, vimentin, and EpCAM were indeed highly expressed in the CD133-positive hepatoma cells. CONCLUSIONS: These findings confirmed that mass spectrometry-based label-free quantitative proteomics can be used to gain insights into liver cancer stem cells.

Multiple lineages of human breast cancer stem/progenitor cells identified by profiling with stem cell markers.

Heterogeneity of cancer stem/progenitor cells that give rise to different forms of cancer has been well demonstrated for leukemia. However, this fundamental concept has yet to be established for solid tumors including breast cancer. In this communication, we analyzed solid tumor cancer stem cell markers in human breast cancer cell lines and primary specimens using flow cytometry. The stem/progenitor cell properties of different marker expressing-cell populations were further assessed by in vitro soft agar colony formation assay and the ability to form tumors in NOD/SCID mice. We found that the expression of stem cell markers varied greatly among breast cancer cell lines. In MDA-MB-231 cells, PROCR and ESA, instead of the widely used breast cancer stem cell markers CD44(+)/CD24(-/low) and ALDH, could be used to highly enrich cancer stem/progenitor cell populations which exhibited the ability to self renew and divide asymmetrically. Furthermore, the PROCR(+)/ESA(+) cells expressed epithelial-mesenchymal transition markers. PROCR could also be used to enrich cells with colony forming ability from MB-361 cells. Moreover, consistent with the marker profiling using cell lines, the expression of stem cell markers differed greatly among primary tumors. There was an association between metastasis status and a high prevalence of certain markers including CD44(+)/CD24(-/low), ESA(+), CD133(+), CXCR4(+) and PROCR(+) in primary tumor cells. Taken together, these results suggest that similar to leukemia, several stem/progenitor cell-like subpopulations can exist in breast cancer.

Porphyrin homeostasis maintained by ABCG2 regulates self-renewal of embryonic stem cells.

BACKGROUND: Under appropriate culture conditions, undifferentiated embryonic stem (ES) cells can undergo multiple self-renewal cycles without loss of pluripotency suggesting they must be equipped with specific defense mechanisms to ensure sufficient genetic stability during self-renewal expansion. The ATP binding cassette transporter ABCG2 is expressed in a wide variety of somatic and embryonic stem cells. However, whether it plays an important role in stem cell maintenance remains to be defined. METHODOLOGY/PRINCIPAL FINDINGS: Here we provide evidence to show that an increase in the level of ABCG2 was observed accompanied by ES colony expansion and then were followed by decreases in the level of protoporphyrin IX (PPIX) indicating that ABCG2 plays a role in maintaining porphyrin homoeostasis. RNA-interference mediated inhibition of ABCG2 as well as functional blockage of ABCG2 transporter with fumitremorgin C (FTC), a specific and potent inhibitor of ABCG2, not only elevated the cellular level of PPIX, but also arrest the cell cycle and reduced expression of the pluripotent gene Nanog. Overexpression of ABCG2 in ES cells was able to counteract the increase of endogenous PPIX induced by treatment with 5-Aminolevulinic acid suggesting ABCG2 played a direct role in removal of PPIX from ES cells. We also found that excess PPIX in ES cells led to elevated levels of reactive oxygen species which in turn triggered DNA damage signals as indicated by increased levels of gammaH2AX and phosphorylated p53. The increased level of p53 reduced Nanog expression because RNA- interference mediated inhibition of p53 was able to prevent the downregulation of Nanog induced by FTC treatment. CONCLUSIONS/SIGNIFICANCE: The present work demonstrated that ABCG2 protects ES cells from PPIX accumulation during colony expansion, and that p53 and gammaH2AX acts as a downstream checkpoint of ABCG2-dependent defense machinery in order to maintain the self-renewal of ES cells.