[Investigation and analysis of underground noise in Sichuan coal mines].

Objective: To understand the harm degree of underground noise and provide basis for noise control. Methods: In November 2019, 13 typical coal mines in Sichuan Province were selected as the research objects, and a total of 1203 sites and 609 jobs of noise exposure were investigated. Results: The noise intensity P75 >80 dB (A) was measured. The noise intensity of the inspection place of the air compressor is >86 dB (A) , the noise intensity of the inspection place of the gas drainage and the operation place of the main fan is between 80-85 dB (A) . Conclusion: Besides the harm of dust, noise exposure should also be paid attention to, and the measures of sound absorption and sound insulation should be taken or personal protection should be strengthened.

[Study on the value of serum des-γ-carboxy prothrombin in predicting hepatocellular carcinoma recurrence after liver transplantation].

Objective: To explore the clinical value of serum des-γ-carboxy prothrombin (DCP) in predicting hepatocellular carcinoma recurrence after liver transplantation. Methods: A total of 115 cases with hepatocellular carcinoma who underwent liver transplantation in Zhongshan Hospital Affiliated to Fudan University from October 2016 to December 2018 were retrospectively analyzed. Receiver operating characteristic curve analysis, Mann-Whitney U test, Kaplan-Meier method, Log-Rank test, χ2 test, univariate and multivariate Cox regression analysis and other statistical methods were used to explore the value of DCP in predicting tumor recurrence after liver transplantation and its correlation with clinicopathological characteristics. Results: The preoperative serum DCP level in recurrent population after liver transplantation was significantly higher than that in non-recurrent population (P < 0.001). The optimal cut-off value of preoperative DCP for predicting recurrence was 200mAU/ml with the use of receiver operating characteristic curve. The sensitivity, specificity, Youden's index and the receiver operating characteristic curve was 87.90%, 57.30%, 0.452, and 0.726, respectively. Survival analysis results grouped by this cut-off value showed that patients with preoperative DCP ≥200mAU/ml had a higher probability of recurrence (P < 0.001). Further, subgroup survival analysis showed that patients with preoperative DCP≥200 mAU/ ml had a higher probability of recurrence than other cases of alpha-fetoprotein negative subgroup, cumulative tumor diameter ≤ 9 cm subgroup and Milan criteria subgroup (P < 0.05). Cox regression analysis showed that preoperative DCP≥200 mAU/ ml (P = 0.017) and cumulative tumor diameter > 9 cm (P = 0.014) was an independent risk factor for recurrence after liver transplantation. χ (2) test results showed that preoperative serum DCP level was correlated with gender, serum gamma glutamyltransferase level, serum alpha fetoprotein level, cumulative tumor diameter, vascular invasion, tumor differentiation and liver cancer transplant criteria (P < 0.05). Conclusion: Preoperative serum DCP can be used as a supplement to the existing liver cancer transplant criteria to predict hepatocellular carcinoma recurrence after liver transplantation. In addition, the accurate screening of patients with low risk of HCC recurrence after liver transplantation can improve the prognosis and efficacy of liver transplant patients.

[Focus on the controversial problems of orbital decompression in the treatment of dysthyroid optic neuropathy].

Dysthyroid optic neuropathy (DON) is the most common cause of visual loss in thyroid associated ophthalmopathy, for which steroid pulse therapy and orbital decompression are the common treatments currently. While steroid pulse therapy was recommended as the first-choice for DON, orbital decompression remained as an indispensable therapy although controversies on such treatment still exist. The mechanism, approaches and outcomes of orbital decompression, as well as its advantages and disadvantages over steroid pulse therapy were retrospectively reviewed, and it was suggested that the combination therapy of steroid pulse therapy and surgical decompression might be the better choice for the treatment of DON. (Chin J Ophthalmol, 2018, 54: 488-490).

Synthesis, dual-nonlinear magnetic resonance and microwave absorption properties of nanosheet hierarchical cobalt particles.

In this paper, we report the synthesis of three kinds of novel nanosheet hierarchical cobalt particles by adjusting the [C4H4O6](2-)/Co(2+) ratio through a liquid reduction method. We investigated the electromagnetic properties of the cobalt particles in detail over the microwave frequency range of 1-18 GHz. The results show that the real part of the permittivity decreases and the imaginary part of the permeability increases with an increase in the [C4H4O6](2-)/Co(2+) ratio. The permeability displays two resonance peaks over the frequency range. The cobalt particles with [C4H4O6](2-)/Co(2+) = 6 have a maximum reflection loss of -48.03 dB at 13.61 GHz, and the effective absorption bandwidth (RL ≤ -10 dB) is 6.76 GHz corresponding to a thickness of 1.7 mm. Considering the impedance matching and attenuation based on the electromagnetic parameters, we designed a way to obtain cobalt particles with excellent microwave absorption properties by decreasing the real part of permittivity and increasing the imaginary part of permeability.

Non-vitamin K antagonist oral anticoagulants in cardiovascular disease management: evidence and unanswered questions.

WHAT IS KNOWN AND OBJECTIVE: Anticoagulation is important in the management of cardiovascular disorders; however, traditional anticoagulants such as heparins and vitamin K antagonists (VKAs) have limitations, including parenteral administration with the former and the need for coagulation monitoring and dose adjustments with the latter. Three non-VKA oral anticoagulants (OACs), dabigatran, rivaroxaban and apixaban, are available for the prevention of stroke in patients with atrial fibrillation (AF) and may change clinical practice. This article reviews current knowledge and important unanswered questions on the use of these agents in patients with cardiovascular disease. METHODS: A literature search was performed using PubMed and the search terms dabigatran, rivaroxaban, apixaban, AF and acute coronary syndrome (ACS). Peer-reviewed, published clinical trials, review articles, relevant treatment guidelines and prescribing information documents were identified and reviewed for relevance. RESULTS AND DISCUSSION: Dabigatran is an oral direct thrombin inhibitor; rivaroxaban and apixaban are oral direct Factor Xa inhibitors. These agents have a quicker onset and offset of action, more predictable pharmacokinetic and pharmacodynamic profiles, and fewer drug-drug interactions than VKAs, allowing use of fixed doses. For the prevention of stroke in patients with AF, the non-VKA OACs were either non-inferior or superior to warfarin with similar or improved bleeding profiles, particularly with respect to reductions in intracranial haemorrhage. In patients with ACS receiving dual antiplatelet therapy, the addition of rivaroxaban significantly reduced the rate of death from cardiovascular causes, myocardial infarction or stroke without increasing fatal bleeding, but led to higher rates of major bleeding. Dose reductions with non-VKA OACs are mandated in certain circumstances in patients with AF, such as moderate renal impairment. Contraindications include creatinine clearance <15 mL/min (<30 mL/min for dabigatran in Europe and Canada) and moderate or severe hepatic impairment, but patients can be transitioned to other anticoagulants if appropriate. It is unknown which non-VKA OAC is optimal for stroke prevention in patients with AF, although factors such as co-medications (e.g. dabigatran may be preferred if a patient is taking a co-medication that is a strong cytochrome P450 3A4 inhibitor) and renal function (rivaroxaban and apixaban depend less on renal clearance than dabigatran) will be important for individual patients. Addition of rivaroxaban to antiplatelet therapy for prevention of recurrent events in patients with recent ACS is approved in Europe for patients at the highest risk (with elevated cardiac biomarkers) and must take into account the increased risk of major bleeding. Although routine coagulation monitoring is not required, an understanding of which assays are appropriate for each non-VKA OAC and how they are affected is important. In a bleeding emergency, non-specific prohaemostatic agents are suggested to reverse the action of the non-VKA OACs, but more clinical data are needed. WHAT IS NEW AND CONCLUSION: Non-VKA OACs provide similar or improved efficacy and, on current evidence, improved safety. They provide greater convenience, compared with traditional anticoagulants for the prevention of stroke in patients with AF. Rivaroxaban may be of benefit to selected high-risk patients with ACS. Selection of the most appropriate non-VKA OAC will depend on individual patient factors.

COVID-19 and bone health.

A few patients who have recovered from COVID-19 develop persistent or new symptoms that last for weeks or months; this is called "long COVID" or "post-COVID-19 syndrome." Over time, awareness of the short- and long-term consequences of COVID-19 has increased. The pulmonary consequences are now fairly well established, but little is known about the extrapulmonary system of COVID-19, particularly its effects on bones. Current evidence and reports indicate a direct relationship between SARS-CoV-2 infection and bone health, with SARS-CoV-2 having a significant negative effect on bone health. In this review, we analyzed the impact of SARS-CoV-2 infection on bone health and assessed the impact of COVID-19 on the diagnosis and treatment of osteoporosis.

[Association between a novel regulatory genetic variants and lung cancer risk in Chinese: a two-stage case-control study].

Objective: Regulatory quantitative trait loci (regQTL) theory can help to evaluate the regulation function of single nucleotide polymorphisms (SNPs) on crucial biological signals from a three-dimensional perspective. The aim of this study was to investigate the effect of these regQTL-SNPs on the susceptibility of lung cancer. Methods: Based on the regQTL theory, using the database of identified lung cancer regQTL-SNPs, we screened the SNPs that may function as regQTL in the reported susceptible regions of lung cancer by genome-wide association study(GWAS), and a two-stage case-control study was conducted (screening stage: 2 331 lung cancer cases and 3 077 healthy controls; validation stage: 626 lung cancer cases and 667 healthy controls) to definite the association of related regQTL-SNPs with the susceptibility of lung cancer. Results: A total of 8 regQTL-SNPs were screened in the reported susceptible regions of lung cancer by GWAS. Among which, 3 SNPs were significantly associated with the risk of lung cancer (P<0.05) in the screening stage. Further validation results indicated that the variant T allele of rs6998591 in ADRA1A was significantly associated with increased risk of lung cancer (additive model: OR=1.33, 95%CI:1.01-1.74, P=0.040). In addition, the variant G allele of rs11202916 in ACTA2 was significantly associated with decreased risk of lung cancer (recessive model: OR=0.71, 95%CI:0.52-0.96, P=0.026). Stratified analysis indicated that the variant T allele of rs6998591 significantly increased lung squamous cell carcinoma risk (additive model: OR=1.53, 95%CI: 1.01-2.32, P=0.043), while the variant G allele of rs11202916 significantly decreased lung adenocarcinoma risk (additive model: OR=0.83, 95%CI: 0.69-0.98, P=0.031). Gene-environment interaction analysis indicated that the risk of developing lung cancer increased by 235% in smoking individuals carrying rs6998591 variant T allele compared with those non-smoking individuals carrying no rs6998591 variant T allele(OR=3.35,95%CI:2.10-5.34,P<0.001). Conclusion: There are two regQTL-SNPs that could significantly affect the susceptibility of lung cancer in the GWAS reported susceptible regions of lung cancer.

Spindle dynamics and cell cycle regulation of dynein in the budding yeast, Saccharomyces cerevisiae.

We have used time-lapse digital- and video-enhanced differential interference contrast (DE-DIC, VE-DIC) microscopy to study the role of dynein in spindle and nuclear dynamics in the yeast Saccharomyces cerevisiae. The real-time analysis reveals six stages in the spindle cycle. Anaphase B onset appears marked by a rapid phase of spindle elongation, simultaneous with nuclear migration into the daughter cell. The onset and kinetics of rapid spindle elongation are identical in wild type and dynein mutants. In the absence of dynein the nucleus does not migrate as close to the neck as in wild-type cells and initial spindle elongation is confined primarily to the mother cell. Rapid oscillations of the elongating spindle between the mother and bud are observed in wild-type cells, followed by a slower growth phase until the spindle reaches its maximal length. This stage is protracted in the dynein mutants and devoid of oscillatory motion. Thus dynein is required for rapid penetration of the nucleus into the bud and anaphase B spindle dynamics. Genetic analysis reveals that in the absence of a functional central spindle (ndcl), dynein is essential for chromosome movement into the bud. Immunofluorescent localization of dynein-beta-galactosidase fusion proteins reveals that dynein is associated with spindle pole bodies and the cell cortex: with spindle pole body localization dependent on intact microtubules. A kinetic analysis of nuclear movement also revealed that cytokinesis is delayed until nuclear translocation is completed, indicative of a surveillance pathway monitoring nuclear transit into the bud.

Long-term donor health and its relationship with outcome of allogeneic hematopoietic stem cell transplantation.

Data on long-term follow-up of donors for hematopoietic stem cell transplantation (HSCT) are limited. Donors of 612 adult allogeneic HSCT were studied, at a median of 81 (14-181) months post-HSC donation. Nine donors had severe health problems. Five donors died from aggressive malignancies or terminal illness, at a median of 41 (16-57) months post-donation. Notably, all their recipients had leukemic relapses. In contrast, donors of recipients in remission were all living. This observation might be due to an inherent depressed immunosurveillance in the donors, or selection of donors with suboptimal health for desperate patients with poor risks pre-HSCT.

The solution structure of [d(CGC)r(aaa)d(TTTGCG)](2): hybrid junctions flanked by DNA duplexes.

The solution structure and hydration of the chimeric duplex [d(CGC)r(aaa)d(TTTGCG)](2), in which the central hybrid segment is flanked by DNA duplexes at both ends, was determined using two-dimensional NMR, simulated annealing and restrained molecular dynamics. The solution structure of this chimeric duplex differs from the previously determined X-ray structure of the analogous B-DNA duplex [d(CGCAAATTTGCG)](2)as well as NMR structure of the analogous A-RNA duplex [r(cgcaaauuugcg)](2). Long-lived water molecules with correlation time tau(c)longer than 0.3 ns were found close to the RNA adenine H2 and H1' protons in the hybrid segment. A possible long-lived water molecule was also detected close to the methyl group of 7T in the RNA-DNA junction but not with the other two thymines (8T and 9T). This result correlates with the structural studies that only DNA residue 7T in the RNA-DNA junction adopts an O4'-endo sugar conformation, while the other DNA residues including 3C in the DNA-RNA junction, adopt C1'-exo or C2'-endo conformations. The exchange rates for RNA C2'-OH were found to be approximately 5-20 s(-1). This slow exchange rate may be due to the narrow minor groove width of [d(CGC)r(aaa)d(TTTGCG)](2), which may trap the water molecules and restrict the dynamic motion of hydroxyl protons. The minor groove width of [d(CGC)r(aaa)d(TTTGCG)](2)is wider than its B-DNA analog but narrower than that of the A-RNA analog. It was further confirmed by its titration with the minor groove binding drug distamycin. A possible 2:1 binding mode was found by the titration experiments, suggesting that this chimeric duplex contains a wider minor groove than its B-DNA analog but still narrow enough to hold two distamycin molecules. These distinct structural features and hydration patterns of this chimeric duplex provide a molecular basis for further understanding the structure and recognition of DNA. RNA hybrid and chimeric duplexes.

Conformation of the propeptide domain of factor IX.

The propeptide domain in the precursor forms of blood clotting proteins contains the recognition sequences for gamma-carboxylase. In hemophilia B, several point mutations in this propeptide domain are responsible for the inherited disease. A peptide containing the propeptide sequence of factor IX was synthesized by solid phase methods. Two dimensional 1H-NMR and CD studies indicate that this peptide motif adopts an alpha-helical structure in a 40% trifluoroethanol-containing aqueous solution. The results suggest that the amphipathic alpha-helix within the propeptide domain of factor IX could create a recognition surface for gamma-carboxylase. The influences of mutations and their relationship with the alpha-helical structure are discussed.

DNA sequence GCGAATGAGC containing the human centromere core sequence GAAT forms a self-complementary duplex with sheared G.A pairs in solution.

The DNA sequence dGCGAATGAGC has a well-resolved, two-dimensional nuclear Overhauser (NOESY) spectrum that is suitable for high quality solution structure determination by NMR methods; in solution this sequence forms a stable self-complementary duplex containing sheared G.A base-pairs. A total of 220 distance constraints derived from time-dependent NOE measurements were collected and refined by repeated back-calculation of the NOESY spectra. Distance information from imino proton studies and from exclusive two-dimensional correlated spectroscopy (E. COSY) and/or linewidth analysis was included in the structure calculation using the program DSPACE 4.2, followed by restrained energy minimization with the program DISCOVER using the AMBER force field. The energies of the distance geometry (DG) structures decreased rapidly in the first few cycles and approached -510 +/- 3 kcal after 1000 cycles of conjugate gradient minimization (about 540 kcal lower than in the initial DG structures). All 15 final DG structures converged to a single family of closely related structures with pair-wise r.m.s.d. values of 0.96 +/- 0.34 A, which was further reduced by energy minimization to 0.70 +/- 0.35 A. Rather unusual structural features of the duplex are revealed in the final structures. The results indicate that, in addition to normal sequences with standard base-pairing, unusual nucleic acid structures can also be determined in solution with quite high precision by NMR/distance geometry methods.

Solution structure of [d(GCGTATACGC)]2.

The solution structure of the alternating pyrimidine-purine DNA duplex [d(GCGTATACGC)]2 has been determined using two-dimensional nuclear magnetic resonance techniques and distance geometry methods. Backbone distance constraints derived from experimental nuclear Overhauser enhancement and J-coupling torsion angle constraints were required to adequately define the conformation of the inter-residue backbone linkages and to avoid underwinding of the duplex. The distance geometry structures were further refined by back-calculation of the two-dimensional nuclear Overhauser enhancement spectra to correct spin-diffusion distance errors. Fifteen final structures for [d(GCGTATACGC)]2 were generated from the refined experimental distance bounds. These structures all exhibit fully wound B-form geometry with small penalty values (< 1.5 A) against the distance bounds and small pair-wise root-mean-square deviation values (typically 0.6 A to 1.5 A). The final structures exhibit positive base-pair inclination with respect to the helix axis, a marked alternation in rise and twist, and are shorter and wider than classical fiber B-form DNA. The purines were found to adopt a sugar pucker close to the C-2'-endo conformation while pyrimidine sugars exhibited significantly lower pseudorotation phase angles in the C-1'-exo to C-2'-endo range. The minor groove cross-strand steric clashes at pyrimidine-purine steps that would exist in pure B-DNA are attenuated by an increased rise at these steps (and an increased roll angle at TpA steps). Concomitantly the backbone torsion angles of the pyrimidine moieties have larger gamma values, larger epsilon values, and smaller zeta values than the purines. The structures generated by distance geometry methods were also compared with those obtained from restrained molecular dynamics with empirical force-field potentials. The results indicate that the nuclear magnetic resonance/distance geometry approach alone is capable of elucidating most of the salient structural features of double-stranded helical nucleic acids in solution without resorting to empirical energy potentials and without using any structural assumptions from crystallographic data.

Solution structure of [d(ATGAGCGAATA)]2. Adjacent G:A mismatches stabilized by cross-strand base-stacking and BII phosphate groups.

The solution structure of a rather unusual B-form duplex [d(ATGAGCGAATA)]2 has been determined using two-dimensional nuclear magnetic resonance (2D-NMR) and distance geometry methods. This sequence forms a stable ten base-pair B-form duplex with 3' overhangs and two pairs of adjacent G:A mismatches paired via a sheared hydrogen-bonding scheme. All non-exchangeable protons, including the stereo-specific H-5'S/H-5'R of the 3G and 7G residues, were assigned by 2D-NMR. The phosphorus spectrum was assigned using heteronuclear correlation with H-3' and H-4' reasonances. The complete assignments reveal several unusual nuclear Overhauser enhancements (NOEs) and unusual chemical shifts for the neighboring G:A mismatch pairs and their adjacent nucleotides. Inter-proton distances were derived from time-dependent NOEs and used to generate initial structures, which were further refined by iterative back-calculation of the two-dimensional nuclear Overhauser enhancement spectra; 22 final structures were calculated from the refined distance bounds. All these final structures exhibit fully wound helical structures with small penalty values against the refined distance bounds and small pair-wise root-mean-square deviation values (typically 0.5 A to 0.9 A). The two helical strands exchange base stacking at both of the two G:A mismatch sites, resulting in base stacking down each side rather than down each strand of the twisted duplex. Very large twist angles (77 degrees) were found at the G:A mismatch steps. All the final structures were found to have BII phosphate conformations at the adjacent G:A mismatch sites, consistent with observed downfield 31P chemical shifts and Monte-Carlo conformational search results. Our results support the hypothesis that 31P chemical shifts are related to backbone torsion angles. These BII phosphate conformations in the adjacent G:A mismatch step suggest that hydrogen bonding of the G:A pair G-NH2 to a nearby phosphate oxygen atom is unlikely. The unusual structure of the duplex may be stabilized by strong interstrand base stacking as well as intrastrand stacking, as indicated by excellent base overlap within the mismatch stacks.

Base pairing geometry in GA mismatches depends entirely on the neighboring sequence.

We have synthesized nine self-complementary DNA oligomers containing different flanking sequences adjacent to a pair of contiguous GA mismatches, and have used high resolution nuclear magnetic resonance (n.m.r.) to investigate the GpA phosphodiester backbone conformation and mismatch pairing schemes in these duplexes. We found dramatic effects of the flanking base pair on the hydrogen bonding and backbone conformation, which appear to be coupled. Thus the Ganti-Aanti base pairing scheme in a NAGATN sequence switches to a more stable sheared GA base pairing scheme in a NCGAGN or NTGAAN context, while no duplex is formed (or only GA bulges occur) when NAGATN is changed to NGGACN. Furthermore, the more stable sheared GA pairing in NPyGAPuN sequences is associated with a BII rather than BI backbone conformation for the phosphodiester between the adjacent mismatched GA pairs. The overall stability of these adjacent GA mismatches as measured by imino proton n.m.r. studies is Py-GA-Pu > A-GA-T > G-GA-C.

Hairpin loops consisting of single adenine residues closed by sheared A.A and G.G pairs formed by the DNA triplets AAA and GAG: solution structure of the d(GTACAAAGTAC) hairpin.

The DNA undecamers GTACAAAGTAC (AAA 11-mer) and GTACGAGGTAC (GAG 11-mer) have been studied in solution by high-resolution NMR spectroscopy. Both duplexes form stable hairpins containing single deoxyadenosine loops and stems containing five base-pairs that are closed at the loop end by sheared AxA and GxC pairs, respectively. These molecules thus contain new AAA and GAG loop turn motifs. All protons, including the chiral H5'/H5" protons of the loop residues, were assigned using NOESY, DQF-COSY and heteronuclear 1H-31P COSY experiments. The backbone torsion angles were constrained using experimental data from NOE crosspeaks, three-bond 1H-1H coupling constants and four-bond 1H-31P coupling constants and four-bond 1H-31P coupling constants. The AAA and GAG 11-mers form similar structures in solution. The detailed structure of the AAA 11-mer was determined by the combined use of NMR, distance geometry and energy minimization methods. This structure exhibits good stacking of the loop adenosine base on the closing 5Ax7A sheared pair, with the 6A base stacking on the 5A base and the 6A deoxyribose stacking with the 7A base. All sugars in the AAA 11-mer hairpin adopt the typical DNA C2'-endo conformation and a sharp backbone turn occurs between residues 6A and 7A. This loop turn is brought about mainly by a change in the backbone phosphate torsion angles from zeta(g-) alpha(g-) to zeta(g+) alphat(g+) at the turn. The gamma torsion angle of residue 7A in the closing sheared pair also changes from gauche+ to trans. In Pu1NPu2 loop turns of the GCA, AAA and GAG types, the chemical shift of the H4' proton of the loop deoxyribose depends on the nature of Pu2; this reflects the stacking of the loop sugar on the Pu2 base and the different ring current effects of A or G in this position.

Hydration of [d(CGC)r(aaa)d(TTTGCG)](2).

We have studied the hydration and dynamics of RNA C2'-OH in a DNA. RNA hybrid chimeric duplex [d(CGC)r(aaa)d(TTTGCG)](2). Long-lived water molecules with correlation time tau(c) larger than 0.3 ns were found close to the RNA adenine H2 and H1' protons in the hybrid segment. A possible long-lived water molecule was also detected close to the methyl group of 7T in the RNA-DNA junction but not to the other two thymine bases (8T and 9T). This result correlates with the structural studies that only DNA residue 7T in the RNA-DNA junction adopts an O4'-endo sugar conformation (intermediate between B-form and A-form), while the other DNA residues including 3C in the DNA-RNA junction, adopt C1'-exo or C2'-endo conformations (in the B-form domain). Based on the NOE cross-peak patterns, we have found that RNA C2'-OH tends to orient toward the O3' direction, forming a possible hydrogen bond with the 3'-phosphate group. The exchange rates for RNA C2'-OH were found to be around 5-20 s(-1), compared to 26.7(+/-13.8) s(-1) reported previously for the other DNA.RNA hybrid duplex. This slow exchange rate may be due to the narrow minor groove width of [d(CGC)r(aaa)d(TTTGCG)](2), which may trap the water molecules and restrict the dynamic motion of hydroxyl protons. The distinct hydration patterns of the RNA adenine H2 and H1' protons and the DNA 7T methyl group in the hybrid segment, as well as the orientation and dynamics of the RNA C2'-OH protons, may provide a molecular basis for further understanding the structure and recognition of DNA.RNA hybrid and chimeric duplexes.

PTPN11 mutations in pediatric patients with acute myeloid leukemia: results from the Children's Cancer Group.

The PTPN11 gene encodes SHP-2, a nonreceptor protein tyrosine phosphatase that relays signals from activated growth factor receptors to p21(ras) (Ras) and other signaling molecules. Somatic PTPN11 mutations are common in patients with juvenile myelomonocytic leukemia (JMML) and have been reported in some other hematologic malignancies. We analyzed specimens from 278 pediatric patients with acute myelogenous leukemia (AML) who were enrolled on Children's Cancer Group trials 2941 and 2961 for PTPN11 mutations. Missense mutations of PTPN11 were detected in 11 (4%) of these samples. None of these patients had mutations in NRAS; however, one patient had evidence of a FLT3 alteration. Four of the patients with PTPN11 mutations (36%) were boys with French-American-British (FAB) morphology M5 AML (P=0.012). Patients with mutations also presented with elevated white blood cell counts. There was no difference in clinical outcome for patients with and without PTPN11 mutations. These characteristics identify a subset of pediatric AML with PTPN11 mutations that share clinical and biologic features with JMML.

Stability and peptide binding specificity of Btk SH2 domain: molecular basis for X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is caused by mutations in the Bruton's tyrosine kinase (Btk). The absence of functional Btk leads to failure of B-cell development that incapacitates antibody production in XLA patients leading to recurrent bacterial infections. Btk SH2 domain is essential for phospholipase C-gamma phosphorylation, and mutations in this domain were shown to cause XLA. Recently, the B-cell linker protein (BLNK) was found to interact with the SH2 domain of Btk, and this association is required for the activation of phospholipase C-gamma. However, the molecular basis for the interaction between the Btk SH2 domain and BLNK and the cause of XLA remain unclear. To understand the role of Btk in B-cell development, we have determined the stability and peptide binding affinity of the Btk SH2 domain. Our results indicate that both the structure and stability of Btk SH2 domain closely resemble with other SH2 domains, and it binds with phosphopeptides in the order pYEEI > pYDEP > pYMEM > pYLDL > pYIIP. We expressed the R288Q, R288W, L295P, R307G, R307T, Y334S, Y361C, L369F, and 1370M mutants of the Btk SH2 domain identified from XLA patients and measured their binding affinity with the phosphopeptides. Our studies revealed that mutation of R288 and R307 located in the phosphotyrosine binding site resulted in a more than 200-fold decrease in the peptide binding compared to L295, Y334, Y361, L369, and 1370 mutations in the pY + 3 hydrophobic binding pocket (approximately 3- to 17-folds). Furthermore, mutation of the Tyr residue at the betaD5 position reverses the binding order of Btk SH2 domain to pYIIP > pYLDL > pYDEP > pYMEM > pYEEI. This altered binding behavior of mutant Btk SH2 domain likely leads to XLA.

Molecular cloning of growth hormone-releasing hormone/pituitary adenylyl cyclase-activating polypeptide in the frog Xenopus laevis: brain distribution and regulation after castration.

Pituitary adenylyl cyclase-activating peptide (PACAP) appears to regulate several neuroendocrine functions in the frog, but its messenger RNA (mRNA) structure and brain distribution are unknown. To understand the potential role of PACAP in the male frog hypothalamic-pituitary-gonadal axis, we cloned the frog Xenopus laevis PACAP mRNA and determined its distribution in the brain. We then analyzed the castration-induced alterations of mRNA expression for PACAP and its selective type I receptor (PAC1) in the hypothalamic anterior preoptic area, a region known to regulate reproductive function. The PACAP mRNA encodes a peptide precursor predicted to give rise to both GH-releasing hormone and PACAP. The deduced peptide sequence of PACAP-38 was nearly identical to that of human PACAP with one amino acid substitution. Abundant PACAP mRNA was detected in the brain, but not several other tissues, including the testis. In situ hybridization revealed strong expression of the PACAP gene in the dorsal pallium, ventral hypothalamus, and nuclei of cerebellum. PACAP mRNA signals were weak to moderate in the hypothalamic anterior preoptic area and were absent in the pituitary. Castration induced an increase in the expression of PACAP and PAC1 receptor mRNAs in the hypothalamic anterior preoptic area after 3 days. Replacement with testosterone prevented the castration-induced changes. These results provide a molecular basis for studying the physiological functions of PACAP in frog brain and suggest that PACAP may be involved in the feedback regulation of hypothalamic-pituitary-gonadal axis.