Analytical validation of HER2DX genomic test for early-stage HER2-positive breast cancer.

BACKGROUND: HER2DX, a multianalyte genomic test, has been clinically validated to predict breast cancer recurrence risk (relapse risk score), the probability of achieving pathological complete response post-neoadjuvant therapy (pCR likelihood score), and individual ERBB2 messenger RNA (mRNA) expression levels in patients with early-stage human epidermal growth factor receptor 2 (HER2)-positive breast cancer. This study delves into the comprehensive analysis of HER2DX's analytical performance. MATERIALS AND METHODS: Precision and reproducibility of HER2DX risk, pCR, and ERBB2 mRNA scores were assessed within and between laboratories using formalin-fixed paraffin-embedded (FFPE) tumor tissues and purified RNA. Robustness was appraised by analyzing the impact of tumor cell content and protocol variations including different instruments, reagent lots, and different RNA extraction kits. Variability was evaluated across intratumor biopsies and genomic platforms [RNA sequencing (RNAseq) versus nCounter], and according to protocol variations. RESULTS: Precision analysis of 10 FFPE tumor samples yielded a maximal standard error of 0.94 across HER2DX scores (1-99 scale). High reproducibility of HER2DX scores across 29 FFPE tumors and 20 RNAs between laboratories was evident (correlation coefficients >0.98). The probability of identifying score differences >5 units was ≤5.2%. No significant variability emerged based on platform instruments, reagent lots, RNA extraction kits, or TagSet thaw/freeze cycles. Moreover, HER2DX displayed robustness at low tumor cell content (10%). Intratumor variability across 212 biopsies (106 tumors) was <4.0%. Concordance between HER2DX scores from 30 RNAs on RNAseq and nCounter platforms exceeded 90.0% (Cohen's κ coefficients >0.80). CONCLUSIONS: The HER2DX assay is highly reproducible and robust for the quantification of recurrence risk, pCR likelihood, and ERBB2 mRNA expression in early-stage HER2-positive breast cancer.

Spin-glass polyamorphism induced by a magnetic field in LaMnO3 single crystal.

We present experimental evidence of field-driven transition in spin-glass state, similar to pressure-induced transition between amorphous phases in structural and metallic glasses, attributed to the polyamorphism phenomena. Cusp in temperature dependences of ac magnetic susceptibility of weakly disordered LaMnO3 single crystal is registered below the temperature of magnetic ordering. Frequency dependence of the cusp temperature proves its spin-glass origin. The transition induced by a magnetic field in spin-glass state, is manifested by peculiarity in dependence of cusp temperature on applied magnetic field. Field dependent maximum of heat capacity is observed in the same magnetic field and temperature range.

Calponin-Like Protein from Mussel Smooth Muscle Is a Competitive Inhibitor of Actomyosin ATPase.

The goal of this work was to elucidate the mechanism of inhibition of the actin-activated ATPase of myosin subfragment-1 (S1) by the calponin-like protein from mussel bivalve muscle. The calponin-like protein (Cap) is a 40-kDa actin-binding protein from the bivalve muscle of the mussel Crenomytilus grayanus. Kinetic parameters Vmax and KATPase of actomyosin ATPase in the absence and the presence of Cap were determined to investigate the mechanism of inhibition. It was found that Cap mainly causes increase in KATPase value and to a lesser extent the decrease in Vmax, which indicates that it is most likely a competitive inhibitor of actomyosin ATPase. Analysis of Vmax and KATPase parameters in the presence of tropomyosin revealed that the latter is a noncompetitive inhibitor of the actomyosin ATPase.

[Effect of hypo- and hyper- accentuated NK cell activity on embryo implantation].

NK lymphocytes play an important role in implantation and during development in early pregnancy. Recently, we showed that the proportion of NK that expressed CD69 after incubation with K562 (CD69(stim)) cells reflected the NK population excitation potential. In the present study, we investigated the significance of NK activation levels in predicting the implantation outcome in 84 patients following IVF (in vitro fertilization). Remarkably, the patients with an accentuated increase or a decrease of the levels of NK activity, have unfavourable conditions for implantation (9.1%, 3/13 and 15.1%, 5/25, respectively) compared to the patients with the nominally normal levels (52.2%. 25/46). Therefore, a nominally normal level of the NK activity is an important physiological condition and predictive factor for immune readiness to embryo implantation. This study describes an easy, efficient, sensitive and informative method for measuring NK cell activity that is relevant to clinical trials.

[Modulation of S-1 conformation and inhibition of the skeletal muscle S-1-ATPase by calponin of the mussel].

A novel 40 kDa protein has been detected in native thin filaments from catch muscles of the mussel Crenomytilus grayanus. In this study, using skeletal muscle actin and S-1, we investigated the effects of the mussel 40-kDa actin-binding protein on the acto · S-1 ATPase activity. On increasing the 40-kDa actin-binding protein (CaP-40) concentration, the actin-activated ATPase activity decreased, and was inhibited 80% at a CaP-40 to actin ratio of 0.5. Polarized fluorimetry technique and glycerinated muscle fibers were used to study effects of CaP-40 on the orientation and mobility of fluorescent label 1.5-IAEDANS specifically bound to CyS-707 of myosin subfragment-1 in the absence of nucleotide, and in the presence of MgADP or MgATP. We have concluded that CaP-40 binding to actin affects the strong binding of myosin to actin but has no effect on the weak binding. Thus, the influence of the CaP-40 on the formation of strong actomyosin binding forms A · M and A · M · ADP manifests itself by a decrease in the relative content of myosin cross-bridges strongly bound with actin, which probably results in a decrease in the relative content of "switch on" actin monomers in thin filaments. This suggests that, as calponin CaP-40 selects its target the phase of strong actomyosin binding binding which preceded by a phase generating power stroke.

40-kDa protein from thin filaments of the mussel Crenomytilus grayanus changes the conformation of F-actin during the ATPase cycle.

Polarized fluorimetry was used to study in ghost muscle fibers the influence of a 40-kDa protein from the thin filaments of the mussel Crenomytilus grayanus on conformational changes of F-actin modified by the fluorescent probes 1,5-IAEDANS and FITC-phalloidin during myosin subfragment (S1) binding in the absence of nucleotides and in the presence of MgADP or MgATP. The fluorescence probes were rigidly bound with actin, which made the absorption and emission dipoles of the probes sensitive to changes in the orientation and mobility of both actin monomer and its subdomain-1 in thin filaments of the muscle fiber. On modeling different intermediate states of actomyosin, the orientation and mobility of oscillators of the dyes were changed discretely, which suggests multistep changes in the actin conformation during the cycle of ATP hydrolysis. The 40-kDa protein influenced the orientation and mobility of the fluorescent probes markedly, suppressing changes in their orientation and mobility in the absence of nucleotides and in the presence of MgADP, but enhancing these changes in the presence of MgATP. The calponin-like 40-kDa protein is supposed to prevent formation of the strong binding state of actomyosin in the absence of nucleotides and in the presence of MgADP but to activate formation of this state in the presence of MgATP.

40-kDa actin-binding protein of thin filaments of the mussel Crenomytilus grayanus inhibits the strong bond formation between actin and myosin head during the ATPase cycle.

Mobility and spatial orientation of a novel 40-kDa actin-binding protein from the smooth muscle of the mussel Crenomytilus grayanus was studied by polarized fluorometry. The influence of this protein on orientation and mobility of the myosin heads was investigated during modeling the different stages of the ATPase cycle. The 40-kDa actin-binding protein affected the strong actin-myosin binding. We suggest that the 40-kDa actin-binding protein is involved in regulation of the actin-myosin interaction in the smooth muscle of the mussel.

AC response of 2H-NbSe2 single crystals with electron-irradiation-induced defects.

The generation of defects in NbSe(2) single crystals by electron irradiation has been investigated by a combination of ac susceptibility and structural measurements. Remarkably, thanks to the layered structure of NbSe(2), we show that electronic irradiation cannot only create point defects but also in-plane extended defects, which modify anisotropically the ac response. Indeed, the analysis of the onset of the nonlinear susceptibility response, H(ac)(l)(T), as a function of irradiation dose and field orientation shows a correlated increase in the density of anisotropic defects induced by electron irradiation. Also, we measured a decrease in the strength of the pinning (Labusch) constant α(L) accounting for elastic vortex oscillations within the linear Campbell regime for high-dose-irradiated samples in a transverse field, again compatible with the presence of planar defects hindering vortex pinning. X-ray powder diffraction and TEM electron diffraction measurements suggest these in-plane defects may result from the rupture of Se-Se bonds and the formation of nanorods and nanowires by NbSe(2) sheet rolling.

(93)Nb NMR study of the charge density wave state in NbSe(2).

(93)Nb NMR studies were carried out for a single crystal of NbSe(2) at 73.328 MHz in the temperature range 9-300 K to investigate the normal and charge density wave (CDW) states. Detailed analysis of the NMR line shape of the central transition using a classical incommensurate model reveals the change in the conduction electron spin dynamics from above T(CDW). An increase of the Knight shift below T(CDW) reflects modification to the uniform part of the conduction electron density of states. As suggested theoretically, the Knight shift distribution is found to be directly proportional to the square of the amplitude of the CDW. The results further indicate an incommensurate CDW state in 2H-NbSe(2). Analysis of the NMR spectra using the McMillan incommensurate model suggests a large value of the discommensuration parameter (γ) which is almost temperature independent, in contrast to the much smaller value previously reported in the case of 2H-TaSe(2).