Rare cells are low-abundance cells in a much larger population of background cells. Conventional benchtop techniques have limited capabilities to isolate and analyze rare cells because of their generally low selectivity and significant sample loss. Recent rapid advances in microfluidics have been providing robust solutions to the challenges in the isolation and analysis of rare cells. In addition to the apparent performance enhancements resulting in higher efficiencies and sensitivity levels, microfluidics provides other advanced features such as simpler handling of small sample volumes and multiplexing capabilities for high-throughput processing. All of these advantages make microfluidics an excellent platform to deal with the transport, isolation, and analysis of rare cells. Various cellular biomarkers, including physical properties, dielectric properties, as well as immunoaffinities, have been explored for isolating rare cells. In this Focus article, we discuss the design considerations of representative microfluidic devices for rare cell isolation and analysis. Examples from recently published works are discussed to highlight the advantages and limitations of the different techniques. Various applications of these techniques are then introduced. Finally, a perspective on the development trends and promising research directions in this field are proposed.
AIM: To analyze tumor regression grade (TRG) for prognosis of locally advanced rectal adenocarcinoma (LARA) treated with preoperative radiotherapy.
METHODS: One hundred and ninety patients with clinical stage II/III LARA were studied. All patients underwent radical surgery (between 2004 and 2010) after 30-Gy/10-fraction preoperative radiotherapy (pre-RT). All 190 patients received a short course of pre-RT and were reassessed for disease recurrence and survival; the slides of surgical specimens were reviewed and classified according to Mandard TRG. We compared patients with good response (Mandard TRG1 or TRG2) vs patients with bad/poor response (Mandard TRG3-5). Outcomes evaluated were 5-year overall survival (OS), 5-year disease-free survival (DFS), and local, distant and mixed recurrence. Fisher’s exact test or χ2 test, log-rank test and proportional hazards regression analysis were used to calculate the probability that Mandard TRG was associated with patient outcomes.
RESULTS: One hundred and sixty-six of 190 patients (87.4%) were identified as Mandard bad responders (TRG3-5). High Mandard grade was correlated with tumor height (41.7% < 6 cm vs 58.3% ≥ 6 cm, P = 0.050), ypT stage (75% ypT0-2 vs 25% ypT3-4, P = 0.000), and ypN stage (75% ypN0 vs 25% ypN1, P = 0.031). In univariate survival analysis, Mandard grade bad responders had significantly worse OS and DFS than good responders (TRG1/2) (OS, 83.1% vs 96.4%, P = 0.000; DFS, 72.3% vs 92.0%, P = 0.002). In multivariate survival analysis, Mandard bad responders had significantly worse DFS than Mandard good responders (DFS 3.8 years (95%CI: 1.2-12.2 years, P = 0.026).
CONCLUSION: Mandard grade good responders had a favorable prognosis. TRG may be a potential predictor for DFS in LARA after pre-RT.
Tumor regression grade; Preoperative radiotherapy; Rectal adenocarcinoma; Disease-free survival
Head and neck squamous cell carcinoma (HNSCC) accounts for more than 5% of all cancers worldwide. The mortality rate of HNSCC has remained unchanged (approximately 50%) over the last few decades. Ubiquitous overexpression of wild type EGFR in many solid tumors has led to the development of EGFR targeted therapies. EGFR can be constitutively activated via several mechanisms including the truncated, EGFR variant III isoform (EGFRvIII). EGFRvIII lacks exons 2–7 and has been reported to be present in up to 20–40% of HNSCC. EGFRvIII has been shown to contribute to cetuximab resistance. The mechanisms leading to EGFRvIII expression in HNSCC are unknown. The present investigation was undertaken to determine the etiology of EGFRvIII in HNSCC.
Materials and Methods
Fixed HNSCC and glioma tissues were analyzed by fluorescence in situ hybridization for EGFR amplification. DNA and RNA from fresh frozen specimens were used to determine the presence of EGFRvIII transcripts and the mechanisms of expression via PCR, RT-PCR and RNA sequencing.
Unlike glioma, EGFRvIII expression in HNSCC did not correlate with EGFR amplification. We found evidence of genomic deletion of the exon 2–7 in 6 of 7 HNSCC cases examined, however, the presence of genomic deletion did not always result in mRNA expression of EGFRvIII. RNA sequencing with automated alignment did not identify EGFRvIII due to microhomology between intron 1 and exon 8. RNA sequencing analyzed by manual alignment methods did not correlate well with RT-PCR and PCR findings.
These findings suggest that genomic deletion as well as additional regulatory mechanisms may contribute to EGFRvIII expression in HNSCC. Further, large scale automated alignment of sequencing are unlikely to identify EGFRvIII and an assay specifically designed to detect EGFRvIII may be necessary to detect this altered form of EGFR in HNSCC tumors.
Liver fibrosis is a hallmark of clonorchiasis suffered by millions people in Eastern Asian countries. Recent studies showed that the activation of TGF-β/Smad signaling pathway can potently regulate the hepatic fibrogenesis including Schistosoma spp. and Echinococcus multilocularis-caused liver fibrosis. However, little is known to date about the expression of transforming growth factor-β (TGF-β) and other molecules in TGF-β/Smad signaling pathway which may play an important role in hepatic fibrosis caused by C. sinensis.
A total of 24 mice were individually infected orally with 45 metacercariae, both experimental mice and mocked-infected control mice were anesthetized at 4 week post-infection (wk p.i.), 8 wk p.i. and 16 wk p.i., respectively. For each time-point, the liver and serum from each animal were collected to analyze histological findings and various fibrotic parameters including TGF-β1, TGF-β receptors and down-stream Smads activation, as well as fibrosis markers expression.
The results showed that collagen deposition indicated by hydroxyproline content and Masson’s trichrome staining was increased gradually with the development of infection. The expression of collagen type α1 (Col1a) mRNA transcripts was steadily increased during the whole infection. The mRNA levels of Smad2, Smad3 as well as the protein of Smad3 in the liver of C. sinensis-infected mice were increased after 4 wk p.i. (P < 0.05, compared with normal control) whereas the TGF-β1, TGF-β type I receptor (TGFβRI) and TGF-β type II receptor (TGFβRII) mRNA expression in C. sinensis-infected mice were higher than those of normal control mice after 8 wk p.i. (P < 0.05). However, the gene expression of Smad4 and Smad7 were peaked at 4 wk p.i. (P < 0.05), and thereafter dropped to the basal level at 8 wk p.i., and 16 wk p.i., respectively. The concentrations of TGF-β1 in serum in the C. sinensis-infected mice at 8 wk p.i. and 16 wk p.i (P < 0.05) were significantly higher than those in the control mice.
The results of the present study indicated for the first time that the activation of TGF-β/Smad signaling pathway might contribute to the synthesis of collagen type I which leads to liver fibrosis caused by C. sinensis.
Clonorchis sinensis; Liver fibrosis; Transforming growth factor-β; Smads
Previous genetic studies on colorectal carcinomas (CRC) have identified multiple somatic mutations in four candidate pathways (TGF-β, Wnt, P53 and RTK-RAS pathways) on populations of European ancestry. However, it is under-studied whether other populations harbor different sets of hot-spot somatic mutations in these pathways and other oncogenes. In this study, to evaluate the mutational spectrum of novel somatic mutations, we assessed 41 pairs of tumor-stroma tissues from Chinese patients with CRC, including 29 colon carcinomas and 12 rectal carcinomas. We designed Illumina Custom Amplicon panel to target 43 genes, including genes in the four candidate pathways, as well as several known oncogenes for other cancers. Candidate mutations were validated by Sanger sequencing, and we further used SIFT and PolyPhen-2 to assess potentially functional mutations. We discovered 3 new somatic mutations in gene APC, TCF7L2, and PIK3CA that had never been reported in the COSMIC or NCI-60 databases. Additionally, we confirmed 6 known somatic mutations in gene SMAD4, APC, FBXW7, BRAF and PTEN in Chinese CRC patients. While most were previously reported in CRC, one mutation in PTEN was reported only in malignant endometrium cancer. Our study confirmed the existence of known somatic mutations in the four candidate pathways for CRC in Chinese patients. We also discovered a number of novel somatic mutations in these pathways, which may have implications for the pathogenesis of CRC.
Light chain amyloidosis is a devastating disease where immunoglobulin light chains form amyloid fibrils, resulting in organ dysfunction and death. Previous studies have shown a direct correlation between the protein thermodynamic stability and the propensity for amyloid formation for some proteins involved in light chain amyloidosis. Here we investigate the effect of somatic mutations on protein stability and in vitro fibril formation of single and double restorative mutants of the protein AL-103 compared to the wild type germline control protein. A scan rate dependence and hysteresis in the thermal unfolding and refolding was observed for all proteins. This indicates that the unfolding/refolding reaction is kinetically determined with different kinetic constants for unfolding and refolding even though the process remains experimentally reversible. Our structural analysis of AL-103 and AL-103 delP95aIns suggests a kinetic coupling of the unfolding/refolding process with cis-trans prolyl isomerization. Our data reveal that the deletion of Proline 95a (AL-103 delP95aIns), which removes the trans cis diproline motif present in the patient protein AL-103, results in a dramatic increment in the thermodynamic stability and a significant delay in fibril formation kinetics with respect to AL-103. Fibril formation is pH dependent; all proteins form fibrils at pH 2; reactions become slower and more stochastic as the pH increases up to pH 7. Based on these results, we propose that in addition to thermodynamic stability, kinetic stability (possibly influenced by the presence of cis Proline 95a), plays a major role in the AL-103 amyloid fibril formation process.
This study aimed to examine the association between negative life events (NLEs) and attempted suicide in rural China.
Six rural counties were selected from disease surveillance points in Shandong province, China. A total of 409 suicide attempters in rural areas between October 1, 2009, and March 31, 2011, and an equal number of matched controls were interviewed. We compared negative life events experienced within 1 month, 1–3 months, 3–6months, and 6–2 months prior to attempted suicide for cases and prior to interview for controls. We used multivariate logistic regression to examine the association between NLEs and attempted suicide.
Suicide attempters experienced more NLEs within the last year prior to suicide attempt than controls prior to interview (83.1% vs. 33.5%). There was a significant dose-response relationship between NLEs experienced within the last year and increased risk of attempted suicide. Timing of NLEs analysis showed that NLEs experienced in the last month and 6–12 months prior to suicide attempt were significantly associated with elevated risk of attempted suicide, even after adjusting for mental disorders and demographic factors. Of NLEs, quarrelling with spouse, quarrelling with other family members, conflicting with friends or neighbors, family financial difficulty, and serious illness were independently related to attempted suicide.
NLEs are significantly associated with increased risk for attempted suicide in rural China. Stress management and intervention may be important to prevent suicidal behavior in rural China.
Site-specific electrochemical deposition is used to prepare polystyrene (PS)-Ag Janus particle arrays with superhydrophobic properties. The analyte molecules can be significantly enriched using the superhydrophobic property of the PS-Ag Janus particle array before SERS detections, enabling an extremely sensitive detection of molecules in a highly diluted solution (e.g., femtomolar level). This superhydrophobic surface enhanced Raman scattering sensing concept described here is of critical significance in biosensing and bioanalysis. Most importantly, the site-specific electrochemical growth method we developed here is a versatile approach that can be used to prepare Janus particle arrays with different properties for various applications.
The viral cell receptors and infection can be blocked by the expression of the viral receptor-binding protein. Thus, the viral cell receptor is an attractive target for anti-viral strategies, and the identification of viral cell receptor is critical for better understanding and controlling viral disease. As a model system for viral entry and anti-retroviral approaches, avian sarcoma/leukosis virus (ASLV, including the A-J ten subgroups) has been studied intensively and many milestone discoveries have been achieved based on work with ASLV. Here, we used a DF1 cell line expressed viral receptor-binding protein to efficiently identify chicken Annexin A2 (chANXA2) as a novel receptor for retrovirus ALV-J (avian leukosis virus subgroup J). Our data demonstrate that antibodies or siRNA to chANXA2 significantly inhibited ALV-J infection and replication, and over-expression of chANXA2 permitted the entry of ALV-J into its non-permissible cells. Our findings have not only identified chANXA2 as a novel biomarker for anti-ALV-J, but also demonstrated that cell lines with the expression of viral receptor-binding protein could be as efficient tools for isolating functional receptors to identify novel anti-viral targets.
The present study aimed to investigate the association between epidermal growth factor receptor (EGFR) gene mutations and excision repair cross-complementing protein 1 (ERCC1) and ribonucleotide reductase subunit M1 (RRM1) mRNA expression in non-small cell lung cancer (NSCLC) tissue. The quantitative polymerase chain reaction was used to detect EGFR mutations, and ERCC1 and RRM1 mRNA expression in 257 cases of NSCLC. In the NSCLC samples the EGFR mutation rate was 49.03% (126/257). The rate was higher in females and non-smoking patients (P<0.05). High expression of ERCC1 mRNA was observed in 47.47% of the samples (122/257), while a high RRM1 mRNA expression was observed in 61.87% of the samples (159/257). In comparison with patients with NSCLC without EGFR mutations, patients with EGFR mutations had significantly lower levels of ERCC1 mRNA expression (P<0.05); however, EGFR mutations and expression levels of RRM1 mRNA were not correlated in NSCLC tissues (P>0.05). In addition, ERCC1 mRNA expression was not correlated with the expression levels of RRM1 mRNA (P>0.05). In conclusion, patients with NSCLC with EGFR mutations tend to have a low expression of ERCC1 mRNA and may potentially benefit from platinum-based chemotherapy.
non-small cell lung cancer; epidermal growth factor receptor; excision repair cross-complementing protein 1; ribonucleotide reductase subunit M1; molecular detection; individualized treatment
Objective. Osteoarthritis (OA) is a complex arthritic condition in which the genetic factor plays a major role. One of the candidate genes of is the ADAM12 gene, but no consistency has been reached till now. This study aims to investigate the potential role of four single nucleotide polymorphisms (SNPs) of the ADAM12 gene in susceptibility to knee OA and its progression in Chinese Han population. Methods. The rs1278279, rs3740199, rs1044122, and rs1871054 polymorphisms were genotyped and compared in a population based cohort consisting of 164 OA subjects and 200 age- and gender-matched controls. Results. The SNP rs1871054 was found with increased risk of OA susceptibility in comparing the genotype frequencies between the case and control groups no matter for which model of comparison (allele level, dominant model, recessive model, and extreme genotype model). Additionally, the SNP rs1871054 was found associated with increased OA severity according to the K/L grade. Conclusion. In summary, we have identified that the rs1871054 variant within the ADAM12 gene is a risk factor for increased osteoarthritis susceptibility and severity.
This study was designed to test a new approach to drug treatment of autism spectrum disorders (ASDs) in the Fragile X (Fmr1) knockout mouse model.
We used behavioral analysis, mass spectrometry, metabolomics, electron microscopy, and western analysis to test the hypothesis that the disturbances in social behavior, novelty preference, metabolism, and synapse structure are treatable with antipurinergic therapy (APT).
Weekly treatment with the purinergic antagonist suramin (20 mg/kg intraperitoneally), started at 9 weeks of age, restored normal social behavior, and improved metabolism, and brain synaptosomal structure. Abnormalities in synaptosomal glutamate, endocannabinoid, purinergic, and IP3 receptor expression, complement C1q, TDP43, and amyloid β precursor protein (APP) were corrected. Comprehensive metabolomic analysis identified 20 biochemical pathways associated with symptom improvements. Seventeen pathways were shared with human ASD, and 11 were shared with the maternal immune activation (MIA) model of ASD. These metabolic pathways were previously identified as functionally related mediators of the evolutionarily conserved cell danger response (CDR).
The data show that antipurinergic therapy improves the multisystem, ASD-like features of both the environmental MIA, and the genetic Fragile X models. These abnormalities appeared to be traceable to mitochondria and regulated by purinergic signaling.
Electronic supplementary material
The online version of this article (doi:10.1186/2040-2392-6-1) contains supplementary material, which is available to authorized users.
Autism spectrum disorders; Purinergic signaling; Antipurinergic therapy (APT); Mitochondria; Metabolism; Metabolomics; Fragile X syndrome; Genetics; Environment; Maternal immune activation (MIA); Cell danger response (CDR)
Viruses that originate in bats may be the most notorious emerging zoonoses that spill over from wildlife into domestic animals and humans. Understanding how these infections filter through ecological systems to cause disease in humans is of profound importance to public health. Transmission of viruses from bats to humans requires a hierarchy of enabling conditions that connect the distribution of reservoir hosts, viral infection within these hosts, and exposure and susceptibility of recipient hosts. For many emerging bat viruses, spillover also requires viral shedding from bats, and survival of the virus in the environment. Focusing on Hendra virus, but also addressing Nipah virus, Ebola virus, Marburg virus and coronaviruses, we delineate this cross-species spillover dynamic from the within-host processes that drive virus excretion to land-use changes that increase interaction among species. We describe how land-use changes may affect co-occurrence and contact between bats and recipient hosts. Two hypotheses may explain temporal and spatial pulses of virus shedding in bat populations: episodic shedding from persistently infected bats or transient epidemics that occur as virus is transmitted among bat populations. Management of livestock also may affect the probability of exposure and disease. Interventions to decrease the probability of virus spillover can be implemented at multiple levels from targeting the reservoir host to managing recipient host exposure and susceptibility.
emerging infectious diseases of bat origin; Hendra virus in flying-foxes; Nipah virus; severe acute respiratory syndrome coronavirus; Ebola virus; Marburg virus
strain-promoted alkyne-azide cycloaddition; sortase A-mediated ligation; one-pot; PEGylation; fluorescent labeling; thrombomodulin
Four experiments investigated the manner in which people use spatial reference directions to organize spatial memories of two conceptually nested layouts. Participants learned directions of 8 remote cities centered to Beijing or Edmonton, where the experiments occurred, using a map or using direct pointing. The map and the environment were aligned, and participants faced north (0°). Participants also learned locations of 7 objects on a table. Participants faced north (0°) during learning but were instructed to learn the layout along the northwest-southeast (45°–225°) axis. Judgments of relative direction (imagine you are standing at X, facing Y, point to Z) were used to determine spatial reference directions in retrieval of bearings between two objects or two cities. The results showed that when the tested bearing and the imagined heading were within an array, participants used 0° as the reference direction in retrieving bearings between cities but used 45°–225° to retrieve bearings between objects. When the tested bearing and the imagined heading were across two arrays, participants used the reference direction of the array from which the tested bearing was. These results indicated that bearings between items within an array were represented only with respect to the reference directions of this array and the relationship between spatial reference directions in these two arrays was also represented.
In recent years, roles of the immune system in immune surveillance of cancer have been explored using a variety of approaches. The roles of the adaptive immune system have been a major emphasis, but increasing evidence supports a role for innate immune effector cells such as natural killer (NK) cells in tumor surveillance. Here, we discuss some of the evidence for roles in tumor surveillance of innate immune cells, particularly NK cells and other immune cells that express germline-encoded receptors that are often labeled NK receptors. The impact of these receptors and the cells that express them on tumor suppression are summarized. We discuss in detail some of the pathways and events in tumor cells that induce or upregulate cell surface expression of the ligands for these receptors, and the logic of how those pathways serve to identify malignant, or potentially malignant cells. How tumors often evade tumor suppression mediated by innate killer cells is another major subject of the review. We end with a discussion of some of the implications of the various findings with respect to possibly therapeutic approaches.
China continues to face challenges in eliminating mother-to-child transmission of human immunodeficiency virus (HIV), syphilis and hepatitis B virus (HBV).
In 2010, a programme that integrated and standardized prevention of mother-to-child transmission (PMTCT) efforts for HIV, syphilis and HBV was implemented in 1156 counties. At participating antenatal care clinics, pregnant women were offered all three tests concurrently and free of charge. Further interventions such as free treatment, prophylaxis and testing for mothers and their children were provided for HIV and syphilis.
China’s national PMTCT HIV programme started in 2003, at which time there were no national programmes for perinatal syphilis and HBV. In 2009, the rate of maternal-to-child transmission of HIV was 8.1% (57/702). Reported congenital syphilis was 60.8 per 100 000 live births. HBV infection was 7.2% of the overall population infected.
Between 2010 and 2013 the number of pregnant women attending antenatal care clinics with integrated PMTCT services increased from 5.5 million to 13.1 million. In 2013, 12.7 million pregnant women were tested for HIV, 12.6 million for syphilis and 12.7 million for HBV. Mother-to-child transmission of HIV fell to 6.7% in 2013. Data on syphilis transmission are not yet available.
Integrated PMTCT services proved to be feasible and effective, and they are now part of the routine maternal and child health services provided to infected women. The services are provided through a collaboration between maternal and child health clinics, the national and local Centers for Disease Control and Prevention, and general hospitals.
We present a quantitative analysis of the electron transfer between single gold nanorods and monolayer graphene under no electrical bias. Using single particle dark-field scattering and photoluminescence spectroscopy to access the homogenous linewidth, we observe broadening of the surface plasmon resonance for gold nanorods on graphene compared to nanorods on a quartz substrate. Because of the absence of spectral plasmon shifts, dielectric interactions between the gold nanorods and graphene are not important and we instead assign the plasmon damping to charge transfer between plasmon-generated hot electrons and the graphene that acts as an efficient acceptor. Analysis of the plasmon linewidth yields an average electron transfer time of 160 ± 30 fs, which is otherwise difficult to measure directly in the time domain with single particle sensitivity. In comparison to intrinsic hot electron decay and radiative relaxation, we furthermore calculate from the plasmon linewidth that charge transfer between the gold nanorods and the graphene support occurs with an efficiency of ~ 10%. Our results are important for future applications of light harvesting with metal nanoparticle plasmons and efficient hot electron acceptors as well as for understanding hot electron transfer in plasmon-assisted chemical reactions.
Plasmon damping; hot electrons; one-photon photoluminescence; single particle spectroscopy; surface plasmon resonance; graphene; plasmon linewidth
Toxoplasmosis caused by the intracellular parasite Toxoplasma gondii (T. gondii) is a global epidemic parasitic disease. DNA vaccines play an important role in preventing the spread of toxoplasmosis. SAG family genes encoding particular surface proteins of T. gondii are the best candidates of DNA vaccine. As a member of SAG family genes, SAG5 gene has been proved to have better antigenic than SAG1. In addition, alpha-Galactosylceramide (α-GalCer) was used to be an adjuvant in malaria vaccine and received positive results. In this study, the effect of the DNA vaccine enhanced by α-GalCer was evaluated by immunizing BALB/c mice.
In the present study, SAG5D gene of T. gondii was cloned, sequenced, and biologically characterized. BALB/c mice were randomly divided into five groups, including three experimental groups (pEGFP-C1-SAG5D, α-GalCer and α-GalCer/pEGFP-C1-SAG5D) and two control groups (PBS and pEGFP-C1), and were immunized intramuscularly three times. The levels of IgG antibodies and cytokine productions in mouse sera were determined by enzyme-linked immunosorbent assays (ELISA). Two weeks after the last immunization, all mice were challenged intraperitoneally with 1 × 104 tachyzoites of T. gondii and the survival time of mice was recorded.
A significant level of increase of IgG response against the soluble tachyzoite antigens (STAg) was detected by ELISA in experimental group. It revealed relatively high level of IFN-γ production by the spleen cells. There were higher productions of interleukin-4 (IL-4) in α-GalCer treated groups compared to control groups. Challenge experiment showed a longer survival period (11 days compared with 5 days in control) in SAG5D DNA vaccinated mice was found after a lethal challenge with T. gondii RH strain.
The present study suggested that T. gondii SAG5D was a novel and positive DNA vaccine candidate against toxoplasmosis. In addition, the adjuvant (α-GalCer) enhanced the body’s cellular immune response and prolonged the survival time of mice after challenge.
Primary involvement of the larynx in non-Hodgkin’s lymphoma (NHL) is rare. Early symptoms are non-specific and thus, it is difficult to diagnose. In the present study, the case of a 52 year-old male with hoarseness due to diffuse T-cell lymphoma as the first manifestation of precursor T-cell acute lymphoblastic leukemia is presented. Subsequent to treatment with three cycles of the etoposide and cytosine arabinoside (EA) and one cycle of the EA+L-asp chemotherapy regimens, the patient achieved complete remission. A series of consolidation therapy courses were performed subsequently. At present, the patient remains disease-free, indicating that the treatment was effective. Primary involvement of the larynx in NHL is rare. Symptoms in the early stage are subtle and non-specific and thus, diagnosis is difficult to establish. This type of tumor requires special diagnostic and therapeutic attention.
laryngeal lymphoma; primary; leukemia; non-Hodgkin’s lymphoma
To compare in young and old rats longitudinal measurements of retinal nerve fiber layer thickness (RNFLT) and axonal transport 3-weeks after chronic IOP elevation.
IOP was elevated unilaterally in 2- and 9.5-month-old Brown-Norway rats by intracameral injections of magnetic microbeads. RNFLT was measured by spectral domain optical coherence tomography. Anterograde axonal transport was assessed from confocal scanning laser ophthalmolscopy of superior colliculi (SC) after bilateral intravitreal injections of cholera toxin-B-488. Optic nerve sections were graded for damage.
Mean IOP was elevated in both groups (young 37, old 38 mmHg, p = 0.95). RNFL in young rats exhibited 10% thickening at 1-week (50.9±8.1 µm, p<0.05) vs. baseline (46.4±2.4 µm), then 7% thinning at 2-weeks (43.0±7.2 µm, p>0.05) and 3-weeks (43.5±4.4 µm, p>0.05), representing 20% loss of dynamic range. RNFLT in old rats showed no significant change at 1-week (44.9±4.1 µm) vs. baseline (49.2±5.3 µm), but progression to 22% thinning at 2-weeks (38.0±3.7 µm, p<0.01) and 3-weeks (40.0±6.6 µm, p<0.05), representing 59% loss of dynamic range. Relative SC fluorescence intensity was reduced in both groups (p<0.001), representing 77–80% loss of dynamic range and a severe transport deficit. Optic nerves showed 75–95% damage (p<0.001). There was greater RNFL thinning in old rats (p<0.05), despite equivalent IOP insult, transport deficit and nerve damage between age groups (all p>0.05).
Chronic IOP elevation resulted in severely disrupted axonal transport and optic nerve axon damage in all rats, associated with mild RNFL loss in young rats but a moderate RNFL loss in old rats despite the similar IOP insult. Hence, the glaucomatous injury response within the RNFL depends on age.
BACKGROUND: Epidermal growth factor receptor (EGFR) mutation status is crucial in treatment selection for non–small cell lung cancer (NSCLC) patients; however, the detection materials’ availability remains challenging in clinical practice. In this study, we collected surgical resection tissues, lymph node biopsy, and cytological samples for EGFR mutation testing and investigated the associations between gene mutation and clinical characteristics. METHODS: Two hundred and seventy-six NSCLC adenocarcinoma specimens were collected, and highly sensitive amplification refractory mutation system method was implemented for EGFR mutation detection, with clinicopathologic characteristics involved in the final analysis. RESULTS: In the total of 276 samples, 96% (265/276) of tumors obtained evaluable EGFR mutation status, the frequency of mutation was 55.8% (148/265) in all specimens, and three different type samples shared a comparable successful testing rate: 97.4% (38/39) in surgical tumor tissues, 100% (108/108) in lymph node biopsy samples, and 92.2% (119/129) in cytological samples. EGFR mutation was significantly associated with sex, smoking history, lymph node metastasis status (N stage), primary tumor size, testing tissues origin, and sample type (P < .05). Multivariate analysis reconfirmed that smoking history and primary tumor size shared significant correlation with EGFR mutation after adjustment. CONCLUSIONS: Both lymph node biopsy and cytological samples were suitable surrogates for EGFR mutation detection in NSCLC compared with tumor tissues, gene status should be detected widely considering the high EGFR mutation rate, and nonsmoking history together with smaller primary tumor size was an independent indicator of EGFR mutation status.
Dysfunction of neuromuscular junction (NMJ) is involved in a wide range of muscular diseases. The development of neuromuscular junction (NMJ) through which skeletal muscle is innervated requires the functional modulation of AchR clustering on myofibers. However, studies on AchR clustering in vitro are mostly done on monolayer muscle cell culture, which lacks a three-dimensional structure, a prominent limitation of the 2D system. To enable a better understanding of the structure-function correlation underlying skeletal muscle innervation, a muscle system with a well-defined geometry mimicking the in vivo muscular setting is needed. Here, we report a 3D bio-artificial muscle (BAM) bioengineered from GFP-transduced C3H murine myoblasts as a novel in vitro tissue-based model for muscle innervation studies. Our cell biological and molecular analysis showed that this BAM is structurally similar to in vivo muscle tissue and can reach the perinatal differentiation stage, higher than does 2D culture. Effective clustering and morphlogical maturation of AchRs on BAMs induced by agrin and laminin indicates the functional activity and plasticity of this BAM system toward innervation. Taken together, our results show that the BAM provides a favorable 3D environment that at least partially recapitulates real physiological skeletal muscle with regard to innervation. With a convenience of fabrication and manipulation, this 3D in vitro system offers a novel model for studying mechanisms underlying skeletal muscle innervation and testing therapeutic strategies for relevant nervous and muscular diseases.
Neuromuscular junction (NMJ); Nicotinic acetylcholine receptor (AchR); Bio-artificial muscle (BAM); Skeletal muscle; Agrin; Laminin
The NKG2D activating receptor has been implicated in numerous autoimmune diseases. We tested the role of NKG2D in models of autoimmunity and inflammation using NKG2D knockout mice and antibody blockade experiments. The severity of experimental autoimmune encephalitis (EAE) was decreased in NKG2D-deficient mice when the disease was induced with a limiting antigen dose, but unchanged with an optimal antigen dose. Surprisingly, however, NKG2D deficiency had no detectable effect in several other models, including two models of type 1 diabetes, and a model of intestinal inflammation induced by poly(I:C). NKG2D antibody blockade in normal mice also failed to inhibit disease in the NOD diabetes model or the intestinal inflammation model. Published evidence using NKG2D knockout mice demonstrated a role for NKG2D in mouse models of atherosclerosis and liver inflammation, as well as in chronic obstructive pulmonary disease. Therefore, our results suggest that NKG2D plays selective roles in inflammatory diseases.
NKG2D; NK cells; EAE; NOD; type 1 diabetes; intestinal inflammation
Rectal pre-exposure prophylaxis (PrEP) will be a critical component of HIV prevention products due to the prevalence of unprotected receptive anal intercourse among men who have sex with men and heterosexual couples. Given the biological considerations of this compartment and the complexity of HIV infection, design of a successful rectal microbicide product faces a number of challenges. Important information is being compiled to begin to address deficits in knowledge toward design of rectal PrEP products for men and women. Aspects of formulation development and preclinical and clinical evaluation of rectal products studied to date are summarized in this review. This article is based on a presentation at the "Product Development Workshop 2013: HIV and Multipurpose Prevention Technologies," held in Arlington, Virginia on February 21–22, 2013. It forms part of a special supplement to Antiviral Research.
Rectal pre-exposure prophylaxis; microbicides; HIV prevention