Clinical value of cell counts and indices in testicular fine needle aspiration cytology in primary infertility: diagnostic performance compared with histology

OBJECTIVE: To compare the diagnostic value of testicular fine needle aspiration (FNA) cytology with that of open biopsy in primary infertility and nonobstructive azospermia or severe oligozoospermia, to evaluate the reliability of percentage cell counts and cell indices. STUDY DESIGN: Thirty patients (21 azospermic and 9 severe oligozoospermic) who had samples for testicular FNA obtained from both testis (mean age = 28.7) and open biopsy were included in the prospective study. Primary infertility, history, complete physical examination, hormonal assay and testicular ultrasound data were evaluated. One case was excluded because of an unsatisfactory result in aspiration cytology. The percentage population of Sertoli cells and spermatogenetic cells, in addition to spermatic index, sertoli cell index and sperm-Sertoli cell indexes, was calculated. The statistical analysis was determined using the paired t test. RESULTS: Progressively increasing values of the Sertoli cell index and progressively decreasing values of the sperm--Sertoli cell index were seen in maturation arrest, hypospermatogenesis and Sertoli cell-only syndrome. The difference between mean counts and indices in normal spermatogenesis and other histologic categories was statistically significant (p < 0.05). CONCLUSION: Percentage cell counts and cell indices in testicular FNA significantly correlate with histological categories. In primary male infertility, testicular FNA can be performed instead of open biopsy.     

An eIF4E-binding protein regulates katanin protein levels in C. elegans embryos

In Caenorhabditis elegans, the MEI-1–katanin microtubule-severing complex is required for meiosis, but must be down-regulated during the transition to embryogenesis to prevent defects in mitosis. A cullin-dependent degradation pathway for MEI-1 protein has been well documented. In this paper, we report that translational repression may also play a role in MEI-1 down-regulation. Reduction of spn-2 function results in spindle orientation defects due to ectopic MEI-1 expression during embryonic mitosis. MEL-26, which is both required for MEI-1 degradation and is itself a target of the cullin degradation pathway, is present at normal levels in spn-2 mutant embryos, suggesting that the degradation pathway is functional. Cloning of spn-2 reveals that it encodes an eIF4E-binding protein that localizes to the cytoplasm and to ribonucleoprotein particles called P granules. SPN-2 binds to the RNA-binding protein OMA-1, which in turn binds to the mei-1 3′ untranslated region. Thus, our results suggest that SPN-2 functions as an eIF4E-binding protein to negatively regulate translation of mei-1.     

TPC2 Proteins Mediate Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)- and Agonist-evoked Contractions of Smooth Muscle

Agonists such as those acting at muscarinic receptors are thought to induce contraction of smooth muscle primarily through inositol 1,4,5-trisphosphate production and release of Ca²⁺ from sarcoplasmic reticulum. However, the additional Ca²⁺-mobilizing messengers cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) may also be involved in this process, the former acting on the sarcoplasmic reticulum, the latter acting on lysosome-related organelles. In this study, we provide the first systematic analysis of the capacity of inositol 1,4,5-trisphosphate, cADPR, and NAADP to cause contraction in smooth muscle. Using permeabilized guinea pig detrusor and taenia caecum, we show that all three Ca²⁺-mobilizing messengers cause contractions in both types of smooth muscle. We demonstrate that cADPR and NAADP play differential roles in mediating contraction in response to muscarinic receptor activation, with a sizeable role for NAADP and acidic calcium stores in detrusor muscle but not in taenia caecum, underscoring the heterogeneity of smooth muscle signal transduction systems. Two-pore channel proteins (TPCs) have recently been shown to be key components of the NAADP receptor. We show that contractile responses to NAADP were completely abolished, and agonist-evoked contractions were reduced and now became independent of acidic calcium stores in Tpcn2^−/− mouse detrusor smooth muscle. Our findings provide the first evidence that TPC proteins mediate a key NAADP-regulated tissue response brought about by agonist activation of a cell surface receptor.     

SYBR green dye-based probe-free SNP genotyping: Introduction of T-Plex real-time PCR assay

Single-nucleotide polymorphism (SNP) genotyping is widely used in genetic association studies to characterize genetic factors underlying inherited traits. Despite many recent advances in high-throughput SNP genotyping, inexpensive and flexible methods with reasonable throughput levels are still needed. Real-time PCR methods for discovering and genotyping SNPs are becoming increasingly important in various fields of biology. In this study, we introduce a new, single-tube strategy that combines the tetra-primer ARMS PCR assay, SYBR Green I-based real-time PCR, and melting-point analysis with primer design strategies to detect the SNP of interest. This assay, T-Plex real-time PCR, is based on the T_m discrimination of the amplified allele-specific amplicons in a single tube. The specificity, sensitivity, and robustness of the assay were evaluated for common mutations in the FV, PII, MTHFR, and FGFR3 genes. We believe that T-Plex real-time PCR would be a useful alternative for either individual genotyping requests or large epidemiological studies.     

Sequential binding of αVβ3 and ICAM-1 determines fibrin-mediated melanoma capture and stable adhesion to CD11b/CD18 on neutrophils

Fibrin (Fn) deposition defines several type 1 immune responses, including delayed-type hypersensitivity and autoimmunity in which polymorphonuclear leukocytes (PMNs) are involved. Fn monomer and fibrinogen are multivalent ligands for a variety of cell receptors during cell adhesion. These cell receptors provide critical linkage among thrombosis, inflammation, and cancer metastasis under venous flow conditions. However, the mechanisms of Fn-mediated interactions among immune cells and circulating tumor cells remain elusive. By using a cone-plate viscometer shear assay and dual-color flow cytometry, we demonstrated that soluble fibrinogen and Fn had different abilities to enhance heterotypic aggregation between PMNs and Lu1205 melanoma cells in a shear flow, regulated by thrombin levels. In addition, the involvement of integrin α(v)β(3), ICAM-1, and CD11b/CD18 (Mac-1) in fibrin(ogen)-mediated melanoma-PMN aggregations was explored. Kinetic studies provided evidence that ICAM-1 mediated initial capture of melanoma cells by PMNs, whereas α(v)β(3) played a role in sustained adhesion of the two cell types at a shear rate of 62.5 s(-1). Quantitative analysis of the melanoma-PMN interactions conducted by a parallel-plate flow chamber assay further revealed that at a shear rate of 20 s(-1), α(v)β(3) had enough contact time to form bonds with Mac-1 via Fn, which could not otherwise occur at a shear rate higher than 62.5 s(-1). Our studies have captured a novel finding that leukocytes could be recruited to tumor cells via thrombin-mediated Fn formation within a tumor microenvironment, and α(v)β(3) and ICAM-1 may participate in multistep fibrin(ogen)-mediated melanoma cell adhesion within the circulation.     

Transforming growth factor beta induces sensory neuronal hyperexcitability, and contributes to pancreatic pain and hyperalgesia in rats with chronic pancreatitis

ABSTRACT: BACKGROUND: Transforming growth factor beta (TGFbeta) is upregulated in chronic inflammation, where it plays a key role in wound healing and promoting fibrosis. However, little is known about the peripheral effects of TGFbeta on nociception. METHODS: We tested the in vitro effects of TGFbeta1 on the excitability of dorsal root ganglia (DRG) neurons and the function of potassium (K) channels. We also studied the effects of TGFbeta1 infusion on pain responses to noxious electrical stimulation in healthy rats as well as the effects of neutralization of TGFbeta1 on evoked pain behaviors in a rat model of chronic pancreatitis. RESULTS: Exposure to TGFbeta1 in vitro increased sensory neuronal excitability, decreased voltage-gated A-type K+ currents (IA) and downregulated expression of the Kv1.4 (KCNA4) gene. Further e TGFbeta1 infusion into the naive rat pancreas in vivo induces hyperalgesia and conversely, neutralization of TGFbeta1 attenuates hyperalgesia only in rats with experimental chronic pancreatitis. Paradoxically, TGFbeta1 neutralization in naive rats results in pancreatic hyperalgesia. CONCLUSIONS: TGFbeta1 is an important and complex modulator of sensory neuronal function in chronic inflammation, providing a link between fibrosis and nociception and is a potentially novel target for the treatment of persistent pain associated with chronic pancreatitis.     

Proteomic-based biomarker discovery for development of next generation diagnostics

Applied Microbiology and Biotechnology - In the post-genome age, proteomics is receiving significant attention because they provide an invaluable source of biological structures and functions at...