Platelet glycoprotein Ib beta/IX mediates glycoprotein Ib alpha localization to membrane lipid domain critical for von Willebrand factor interaction at high shear

The localization of the platelet glycoprotein GP Ib-IX complex (GP Ibα, GP Ibβ, and GP IX) to membrane lipid domain, also known as glycosphingolipid-enriched membranes (GEMs or raft) lipid domain, is essential for the GP Ib-IX complex mediated platelet adhesion to von Willebrand factor (vWf) and subsequent platelet activation. To date, the mechanism for the complex association with the GEMs remains unclear. Although the palmitate modifications of GP Ibβ and GP IX were thought to be critical for the complex presence in the GEMs, we found that the removal of the putative palmitoylation sites of GP Ibβ and GP IX had no effects on the localization of the GP Ib-IX complex to the GEMs. Instead, the disruption of GP Ibα disulfide linkage with GP Ibβ markedly decreased the amount of the GEM-associated GP Ibα without altering the GEM association of GP Ibβ and GP IX. Furthermore, partial dissociation with the GEMs greatly inhibited GP Ibα interaction with vWf at high shear instead of in static condition or under low shear stress. Thus, for the first time, we demonstrated that GP Ibβ/GP IX mediates the disulfide-linked GP Ibα localization to the GEMs, which is critical for vWf interaction at high shear.     

Factors Influencing the Degradation of Archival Formalin-Fixed Paraffin-Embedded Tissue Sections

The loss of antigenicity in archival formalin-fixed paraffin-embedded (FFPE) tissue sections negatively affects both diagnostic histopathology and advanced molecular studies. The mechanisms underlying antigenicity loss in FFPE tissues remain unclear. The authors hypothesize that water is a crucial contributor to protein degradation and decrement of immunoreactivity in FFPE tissues. To test their hypothesis, they examined fixation time, processing time, and humidity of storage environment on protein integrity and antigenicity by immunohistochemistry, Western blotting, and protein extraction. This study revealed that inadequate tissue processing, resulting in retention of endogenous water in tissue sections, results in antigen degradation. Exposure to high humidity during storage results in significant protein degradation and reduced immunoreactivity, and the effects of storage humidity are temperature dependent. Slides stored under vacuum with desiccant do not protect against the effects of residual water from inadequate tissue processing. These results support that the presence of water, both endogenously and exogenously, plays a central role in antigenicity loss. Optimal tissue processing is essential. The parameters of optimal storage of unstained slides remain to be defined, as they are directly affected by preanalytic variables. Nevertheless, minimization of exposure to water is required for antigen preservation in FFPE tissue sections. This article contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.     

Lysophosphatidylcholine as an effector of fatty acid-induced insulin resistance

The mechanism of FFA-induced insulin resistance is not fully understood. We have searched for effector molecules(s) in FFA-induced insulin resistance. Palmitic acid (PA) but not oleic acid (OA) induced insulin resistance in L6 myotubes through C-Jun N-terminal kinase (JNK) and insulin receptor substrate 1 (IRS-1) Ser307 phosphorylation. Inhibitors of ceramide synthesis did not block insulin resistance by PA. However, inhibition of the conversion of PA to lysophosphatidylcholine (LPC) by calcium-independent phospholipase A₂ (iPLA₂) inhibitors, such as bromoenol lactone (BEL) or palmitoyl trifluoromethyl ketone (PACOCF₃), prevented insulin resistance by PA. iPLA₂ inhibitors or iPLA₂ small interfering RNA (siRNA) attenuated JNK or IRS-1 Ser307 phosphorylation by PA. PA treatment increased LPC content, which was reversed by iPLA₂ inhibitors or iPLA₂ siRNA. The intracellular DAG level was increased by iPLA₂ inhibitors, despite ameliorated insulin resistance. Pertussis toxin (PTX), which inhibits LPC action through the G-protein coupled receptor (GPCR)/Gα_i, reversed insulin resistance by PA. BEL administration ameliorated insulin resistance and diabetes in db/db mice. JNK and IRS-1Ser307 phosphorylation in the liver and muscle of db/db mice was attenuated by BEL. LPC content was increased in the liver and muscle of db/db mice, which was suppressed by BEL. These findings implicate LPC as an important lipid intermediate that links saturated fatty acids to insulin resistance.     

Systematic blade production at late Lower Paleolithic (400-200 kyr) Qesem Cave, Israel

Qesem Cave is assigned to the Acheulo-Yabrudian cultural complex of the late Lower Paleolithic period. The 7.5 m deep stratigraphic sequence is dated to 400-200 ka (thousands of years ago). It is mostly attributed to the Amudian blade-dominated industry, one of the earliest blade production technologies in the world. In this paper, we present the results of a detailed study of five Amudian assemblages from Qesem Cave and suggest two trajectories for the production of blades at the site. We argue that the reduction sequences of blades at Qesem Cave represent an innovative and straightforward technology aimed at the systemic and serial production of predetermined blanks. We suggest that this predetermined blank technology shows planning and intensity that is not significantly different from Middle Paleolithic Mousterian technological systems. Furthermore, this well-organized serial manufacture of cutting implements mainly for butchering might indicates that a significant change in human behavior had taken place by the late Lower Paleolithic period.     

Positive feedback loops sustain repeating bursts in neuronal circuits

Voluntary movements in animals are often episodic, with abrupt onset and termination. Elevated neuronal excitation is required to drive the neuronal circuits underlying such movements; however, the mechanisms that sustain this increased excitation are largely unknown. In the medicinal leech, an identified cascade of excitation has been traced from mechanosensory neurons to the swim oscillator circuit. Although this cascade explains the initiation of excitatory drive (and hence swim initiation), it cannot account for the prolonged excitation (10–100 s) that underlies swim episodes. We present results of physiological and theoretical investigations into the mechanisms that maintain swimming activity in the leech. Although intrasegmental mechanisms can prolong stimulus-evoked excitation for more than one second, maintained excitation and sustained swimming activity requires chains of several ganglia. Experimental and modeling studies suggest that mutually excitatory intersegmental interactions can drive bouts of swimming activity in leeches. Our model neuronal circuits, which incorporated mutually excitatory neurons whose activity was limited by impulse adaptation, also replicated the following major experimental findings: (1) swimming can be initiated and terminated by a single neuron, (2) swim duration decreases with experimental reduction in nerve cord length, and (3) swim duration decreases as the interval between swim episodes is reduced.     

Structure of a key intermediate of the SMN complex reveals Gemin2's crucial function in snRNP assembly

The SMN complex mediates the assembly of heptameric Sm protein rings on small nuclear RNAs (snRNAs), which are essential for snRNP function. Specific Sm core assembly depends on Sm proteins and snRNA recognition by SMN/Gemin2- and Gemin5-containing subunits, respectively. The mechanism by which the Sm proteins are gathered while preventing illicit Sm assembly on non-snRNAs is unknown. Here, we describe the 2.5?? crystal structure of Gemin2 bound to SmD1/D2/F/E/G pentamer and SMN's Gemin2-binding domain, a key assembly intermediate. Remarkably, through its extended conformation, Gemin2 wraps around the crescent-shaped pentamer, interacting with all five Sm proteins, and gripping its bottom and top sides and outer perimeter. Gemin2 reaches into the RNA-binding pocket, preventing RNA binding. Interestingly, SMN-Gemin2 interaction is abrogated by a spinal muscular atrophy (SMA)-causing mutation in an SMN helix that mediates Gemin2 binding. These findings provide insight into SMN complex assembly and specificity, linking snRNP biogenesis and SMA pathogenesis.     

口腔鳞状细胞癌患者外周血Naa10及淋巴细胞免疫分型的特点及临床意义

目的探讨口腔鳞状细胞癌（OSCC）患者外周血N-α-乙酰基转移酶10 （Naa10）及淋巴细胞免疫分型的特点,并分析其临床意义。 方法选取97例OSCC患者及50名健康体检者,分别纳入患者组、对照组。检测两组受试者外周血Naa10、淋巴细胞免疫分型,并比较不同TNM分期、不同淋巴结转移状态患者外周血Naa10、淋巴细胞免疫分型的差异,总结其特点与临床意义。不同性别人数采用χ²检验,计量资料行正态性检验,满足正态性且组间方差相等则采用t检验,不满足正态性则采用非参数Wilcoxon秩和检验,相关性分析采用Pearson法。 结果患者组血清Naa10为（62.91±17.15）pg/ml,高于对照组的（38.82±9.04）pg/ml,差异有统计学意义（t?= 9.280,P?< 0.05）。患者组CD3⁺、CD4⁺、CD4⁺/CD8⁺、NK细胞百分比分别为（71.63±9.11）﹪、（34.34±4.26）﹪、（1.03±0.28）、（14.23±4.61）﹪,低于对照组的（76.25±2.87）﹪、（45.41±7.08）﹪、（1.77± 0.39）、（19.96±5.13）﹪,CD8⁺、B淋巴细胞百分比分别为（14.23±4.61）﹪、（14.91±3.29）﹪,高于后者的（19.96±5.13）﹪、（9.11±2.60）﹪,差异有统计学意义（P?< 0.05）。TNM分期Ⅰ~Ⅱ期患者42例,其血清Naa10为（64.08±15.26）?pg/?ml,与Ⅲ~Ⅳ期患者的（61.71±13.15）pg/ml比较,差异无统计学意义（t?= 0.820,P?> 0.05）。淋巴结转移者34例,其血清Naa10为（65.15±14.07）pg/ml,与无淋巴结转移者的（61.45±12.66）?pg/?ml比较,差异无统计学意义（t?= 1.321,P?> 0.05）。Ⅰ~Ⅱ期患者CD3⁺、CD4⁺、CD4⁺/CD8⁺、NK细胞百分比低于Ⅲ~Ⅳ期患者,其CD8⁺、B淋巴细胞百分比高于后者,差异有统计学意义（P?< 0.05）。Pearson相关性分析示,外周血Naa10与CD3⁺ （-0.631）、CD4⁺ （-0.529）、CD4⁺/CD8⁺ （-0.587）、NK细胞百分比（-0.603）呈负相关,与CD8⁺ （0.558）、B淋巴细胞百分比（0.670）呈正相关（P?< 0.05）。 结论OSCC患者外周血Naa10明显升高但与TNM分期、淋巴结转移无关;其淋巴细胞免疫分型存在明显改变,且TNM分期的上升、淋巴结转移的发生伴随着CD3⁺、CD4⁺、CD4⁺/?CD8⁺、NK细胞百分比的下降,以及CD8⁺、B淋巴细胞百分比的上升。