Oligonucleotides Containing Consecutive 2′-Deoxy-Isoguanosine Residues: Synthesis,Parallel Duplex Formation and Identification of a d(T4iG4T4) Tetraplex

Abstract

Oligonucleotides containing consecutive 2′-deoxyisoguano-sines were synthesized by using building blocks protected with a diphenylcarb-amoyl residue. Parallel duplexes were formed by iG_d-dC base pairs and a tetraplex of d(T₄iG₄T₄) was identified by ion exchange HPLC.     

A Parallel Stranded Linear DNA Duplex Incorporating dG · dC Base Pairs

Abstract

DNA oligonucleotides with appropriately designed complementary sequences can form a duplex in which the two strands are paired in a parallel orientation and not in the conventional antiparallel double helix of B-DNA. All parallel stranded (ps) molecules reported to date have consisted exclusively of dA · dT base pairs. We have substituted four dA · dT base pairs of a 25-nt parallel stranded linear duplex (ps-D1 · D2) with dG · dC base pairs. The two strands still adopt a duplex structure with the characteristic spectroscopic properties of the ps conformation but with a reduced thermodynamic stability. Thus, the melting temperature of the ps duplex with four dG · dC base pairs (ps-D5 · D6) is 10-16°C lower and the van't Hoff enthalpy difference Δ_vH for the helix-coil transition is reduced by 20% (in NaCl) and 10% (in MgCl₂) compared to that of ps-Dl · D2. Based on energy minimizations of a ps-[d(T₅GA₅) · d(A₅CT₅)] duplex using force field calculations we propose a model for the conformation of a trans dG · dC base pair in a ps helix.     

Fluorescence Properties and Base Pair Stability of Oligonucleotides Containing 8-Aza-7-deaza-2′-deoxyisoinosine or 2′-Deoxyisoinosine

Abstract

The fluorescence and the base pairing properties of 8-aza-7-deaza-2′-deoxyisoinosine (1) are described and compared with those of 2′-deoxyisoinosine (2). The corresponding phosphoramidites (11,12) are synthesized using the diphenyl-carbamoyl (DPC) residue for the 2-oxo group protection. The nucleosides 1 and 2 base pair with 2′-deoxy-5-methylisocytidine in DNA duplexes with antiparallel chain orientation and with 2′-deoxycytidine in a parallel DNA. These base pairs are less stable than the canonical dA-dT pair and that of 2′-deoxyinosine (4) with 2′-deoxycytidine. The fluorescence of the nucleosides 1 and 2 is quenched (～95%) in duplex DNA. The residual fluorescence is used to determine the T_m-values, which are found to be the same as determined UV-spectrophotometrically.     

DNA Sequence-Specific Ligands: XI.* The Synthesis and Binding to DNA of bis-Netropsins with the C-Ends of Their Netropsin Fragments Tethered by Tetra- or Pentamethylene Linkers

Bis-Netropsins with the C-ends of their netropsin fragments tethered via tetra- or pentamethylene linkers and with Gly or L-Lys-Gly residues on their N-ends were synthesized. The footprinting technique was used to study the specificity of bis-netropsin binding to the specially constructed DNA fragments containing various clusters of A · T pairs. It was found that the linker length affects the binding of bis-netropsins, with the tetramethylene linker providing better protection than the pentamethylene linker. It was shown that the newly synthesized bis-netropsins bind tighter to the 5"-A ₄ T ₄-3" sequence, whereas the bis-netropsin with a linker between the netropsin N-ends binds better to 5"-T ₄ A ₄-3" sequences.     

Thermodynamics of DNA Containing Very Stable Chemically Modified Base Pairs

Abstract

DNA chemical modifications caused by the binding of some antitumor drugs give rise to a very strong local stabilization of the double helix. These sites melt at a temperature that is well above the melting temperatures of ordinary AT and GC base pairs. In this work we have examined the melting behavior of DNA containing very stable sites. Analytical expressions were derived and used to evaluate the thermodynamic properties of homopolymers DNA with several different distributions of stable sites. The results were extended to DNA with a heterogeneous sequence of AT and GC base pairs. The results were compared to the melting properties of DNA with ordinary covalent interstrand cross-links. It was found that, as with an ordinary interstrand cross-link, a single strongly stabilized site makes a DNA's melting temperature (T_m ) independent of strand concentration. However in contrast to a DNA with an interstrand cross-link, a strongly stabilized site makes the DNA's T_m independent of DNA length and equal to T _∞, the melting temperature of an infinite length DNA with the same GC-content and without a stabilized site. Moreover, at a temperature where more than 80% of base pairs are melted, the number of ordinary (non-modified) helical base pairs (n) is independent of both the DNA length and the location of the stabilized sites. For this condition, n(T) = (2ω-a) S (1- S ) and S = exp[ΔS(T∞-T)/(RT)] where ω is the number of strongly stabilized sites in the DNA chain, a is the number of DNA ends that contain a stabilized site, and ΔS, T, and R are the base pair entropy change, the temperature, and the universal gas constant per mole. The above expression is valid for a temperature interval that corresponds to n<0.2N for ω=1, and n<0.1N for ω>1, where N is the number of ordinary base pairs in the DNA chain.     

Hysteresis in poly‐2′‐deoxycytidine i‐motif folding is impacted by the method of analysis as well as loop and stem lengths

In DNA, i‐motif (iM) folds occur under slightly acidic conditions when sequences rich in 2′‐deoxycytidine (dC) nucleotides adopt consecutive dC self base pairs. The pH stability of an iM is defined by the midpoint in the pH transition (pH_T) between the folded and unfolded states. Two different experiments to determine pH_T values via circular dichroism (CD) spectroscopy were performed on poly‐dC iMs of length 15, 19, or 23 nucleotides. These experiments demonstrate two points: (1) pH_T values were dependent on the titration experiment performed, and (2) pH‐induced denaturing or annealing processes produced isothermal hysteresis in the pH_T values. These results in tandem with model iMs with judicious mutations of dC to thymidine to favor particular folds found the hysteresis was maximal for the shorter poly‐dC iMs and those with an even number of base pairs, while the hysteresis was minimal for longer poly‐dC iMs and those with an odd number of base pairs. Experiments to follow the iM folding via thermal changes identified thermal hysteresis between the denaturing and annealing cycles. Similar trends were found to those observed in the CD experiments. The results demonstrate that the method of iM analysis can impact the pH_T parameter measured, and hysteresis was observed in the pH_T and T_m values.     

肺保护性通气策略对老年患者腹腔镜结直肠癌手术的氧合功能及血浆炎症介质水平的影响

目的:探讨肺保护通气策略对老年患者行腹腔镜结直肠癌根治术肺部氧合功能及血清炎症介质水平的影响。方法:选择50例行择期腹腔镜结直肠癌根治术老年患者,ASA分级(美国麻醉医师协会体格情况评估分级)Ⅰ~Ⅱ级、年龄≥60岁,采用随机数字表法将其分为两组:VCV组和PCV组。在围术期行全麻机械通气中,VCV组采用容量通气模式,潮气量为8 m L/kg,PCV组采用肺保护通气,潮气量为6 m L/kg及5 cm H2O呼气末正压通气(positive end expiration pressure,PEEP),同时气腹后每30 min给予一次手法肺复张。记录患者气腹前5 min(T0)、气腹后5 min(T1)、气腹后30 min(T2)、气腹后60 min(T3)、气腹后120 min(T4)、气腹停止10 min后(T5)的呼吸力学指标、血流动力学指标于T0、T4、离开苏醒室时抽取血气,计算氧合指数(OI)值,于术前一天、T4、术后一天抽取静脉血,检测血浆CRP、IL-6的值。结果:与VCV组比较,PCV组在T4、T5时刻气道压降低,T4、T5肺顺应性增高(P<0.05)。两组患者血流动力学指标无明显差异。PCV组在离开苏醒室时氧合指数较高(P<0.05);PCV组在术后一天时刻IL-6和CRP值较低(P<0.05)。结论:肺保护性通气策略可以提高老年患者肺部氧合功能,减少炎症介质释放,减轻肺损伤。     

Use of the Free Thyroxine Index to Refine the Lower Limit of a Free Thyroxine Immunoassay for Detection of Secondary Hypothyroidism

ObjectiveTo determine the utility of measuring free T₄ index (FT₄I) in patients with low free T₄ (FT₄) levels using immunoassay and normal thyroid-stimulating hormone for the evaluation of secondary hypothyroidism.MethodsWe performed a retrospective medical chart review of patients seen at a single institution as outpatients who had a simultaneously normal thyroid-stimulating hormone level, low FT₄ level, and any FT₄I measured between June 2014 and October 2016. Demographic, laboratory, and imaging data were collected. Using FT₄I as the reference for diagnosis of hypothyroidism, the sensitivity and specificity of the FT₄ immunoassay’s lower-limit thresholds were determined. Within each threshold group, available brain imaging and biochemical evaluation were categorized according to the presence or absence of pituitary disease.ResultsA total of 155 sets of result pairs (FT₄ and FT₄I) performed on 118 subjects were analyzed. The lower limit of a normal FT₄ level by immunoassay at this institution was 0.93 ng/dL, though all pairs with FT₄ ≥0.89 ng/dL had a normal FT₄I. All pairs with FT₄ ≤0.67 ng/dL had a low FT₄I. No pituitary macroadenomas were identified in any subject, though the rates of pituitary imaging in this patient sample were low.ConclusionPatients with a borderline low FT₄ level by immunoassay often have normal FT₄I. In such patients at our center, significant structural and biochemical pituitary pathology was uncommon.     

盐酸右美托咪定联合瑞芬太尼在骨科手术中的镇静效果及对患者血流动力学和呼吸功能的影响

目的:探讨盐酸右美托咪定联合瑞芬太尼在骨科手术中的镇静效果及对患者血流动力学和呼吸功能的影响。方法:选择2016年3月至2017年5月在我院186例行骨科手术的患者,根据数表法随机分为对照组(93例)和观察组(93例),对照组采用丙泊酚联合瑞芬太尼进行麻醉,观察组采用盐酸右美托咪定联合瑞芬太尼进行麻醉,比较两组用药前(T_1)、联合用药后10 min(T_2)、联合用药后20 min(T_3)、联合用药后30 min(T_4)和患者清醒后(T_5)等时间点的镇静效果以及血流动力学和呼吸功能指标变化,并比较两组不良反应发生率。结果:T_3、T_4时间点两组警觉/镇静(OAA/S)评分低于组内其他时间点,且观察组OAA/S评分低于对照组(P0.05)。两组T_1、T_5时间点呼吸频率(RR)、血氧饱和度(SpO_2)比较差异无统计学意义(P0.05);在T_2、T_3、T_4时间点观察组RR、SpO_2高于对照组(P0.05);T_3、T_4时间点两组的RR水平均低于组内其他时间点(P0.05)。两组各时间点平均动脉压(MAP)、心率(HR)水平比较差异均无统计学意义(P0.05)。观察组呼吸抑制、呼吸道梗塞和恶心呕吐的发生率低于对照组(P0.05)。结论:盐酸右美托咪定联合瑞芬太尼在骨科手术中镇静效果良好,对患者血流动力学的影响较小,呼吸抑制程度轻,安全性好。     

New triterpene oligoglycosides from the Caribbean sponge Erylus formosus

Seven new triterpene glycosides, erylosides R₁ (1), T₁ (3), T₂ (4), T₃ (5), T₄ (6), T₅ (7), and T₆ (8) along with the known formoside (2) were isolated from the sponge Erylus formosus collected along the Caribbean coast of Mexico. Glycoside 1 was determined as a trisaccharide, glycoside 2 as a tetrasaccharide while glycosides 3–8 were hexasaccharide. Their carbohydrate chains were unprecedented and have never been found in oligosaccharides from other biological sources, except Erylus spp. Three carbohydrate chains in the glycosides 3 and 6, 4 and 7, 5 and 8 correspondingly are new. The glycosides 1–5 have penasterol as aglycone while glycosides 6–8 proved to be glycoconjugates of 24-methylene-14-carboxy-lanost-8(9)-en-3β-ol.     

Comparative Studies of the Thermodynamic Stabilities Between Sheared A:G and Watson-Crick A:U(T) Base Pairs in RNA and DNA

Abstract

Thermodynamic parameters for duplex formation were determined from CD melting curves for r(GGACGAGUCC)₂ and d(GGACGAGTCC)₂, both of which form two consecutive ‘sheared’ A:G base pairs at the center [Katahira et al. (1993) Nucleic Acids Res. 21, 5418–5424; Katahira et al., (1994) Nucleic Acids Res. 22, 2752–27591. The parameters were determined also for r(GGACUAGUCC)₂ and d(GGACTAGTCC)₂, where the A:G mismatches are replaced by Watson-Crick A:U(T) base pairs. Thermodynamic properties for duplex formation are compared between the sheared and the Watson-Crick base pairs, and between RNA and DNA. Difference in the thermodynamic stability is analyzed and discussed in terms of enthalpy and entropy changes. The characteristic features in CD spectra of RNA and DNA containing the sheared A:G base pairs are also reported.

     

Design and Synthesis of Triazole-Linked xylo-Nucleoside Dimers

Three triazole-linked nonionic xylo-nucleoside dimers T^L-t-T^xL, T^L-t-A^BzxL and T^L-t-C^BzxL have been synthesized for the first time by Cu(I) catalyzed azide-alkyne [3 + 2] cycloaddition reaction (CuAAC) of 1-(3′-azido-3′-deoxy-2′-O,4′-C-methylene-β-D-ribo-furanosyl)thymine with different alkynes, i.e., 1-(5′-deoxy-5′-C-ethynyl-2′-O,4′-C-methylene-β-D-xylofuranosyl)thymine, 9-(5′-deoxy-5′-C-ethynyl-2′-O,4′-C-methylene-β-D-xylo-furanosyl)-N6-benzoyladenine and 1-(5′-deoxy-5′-C-ethynyl-2′-O,4′-C-methylene-β-D-xylofuranosyl)-N4-benzoylcytosine in 90%–92% yields. Among the two Cu(I) reagents, CuSO₄.5H₂O-sodium ascorbate in THF:^tBuOH:H₂O (1:1:1) and CuBr.SMe₂ in THF used for cycloaddition (click) reaction, the former one was found to be better yielding than the latter one.     

Temperature dependence of the transition enthalpy of core particle DNA and of long DNA

The denaturation of short (145 base pairs) and long (about 8000 base pairs) DNA moelucules has been studied by adiabitic differential microcalorimetry in solutions with different NaCl content. It is found that the enthalpy of denaturation of short DNA is more sensitive to changes in T_m than that of long DNA. A comparison with other data is also given.     

Free energy of imperfect nucleic acid helices. 3. Small internal loops resulting from mismatches

Physical studies of enzymioally synthesized oligoribonucleotides of defined sequence are used to evaluate quantitatively the destabilizing influence of mismatched bases in a double helix. The series (A-)₄G(-C)_n(-U)₄, N = 1 to 6, exist as imperfect dimer helices when N is equal to or less than 4, and as monomolecular hairpin helices when N is 5 and 6. Internal loops become progressively more destabilizing as their size increases from 2 to 4 to 6 nucleotides resulting from 1, 2 and 3 consecutive mismatched base pairs. However, the stability of a helix will generally be greater if a given number of mismatched pairs occur consecutively rather than in isolation from one another.These data may be used for improved calculations of stability of RNA secondary structure, to estimate the frequency of structural fluctuations in a double helix and to assess the stability of modified polynucleotide helices. An unmodified double helix of one million randomly arranged base pairs should contain on the time average approximately 10 G.C and 500 A.U pairs in non-hydrogen bonded, unstacked conformations at 25 °C. Our estimate of the effect of mismatching on T_m values of high polymers is less precise because of the long temperature extrapolation required. However, we estimate that DNA or RNA treated with mutagens which interrupt up to 20% of the nucleotide pairs will show a drop of about 1.2 deg. C in melting temperature with each unit per cent of modification.     

艾司氯胺酮联合舒芬太尼对剖宫产围手术期镇痛、应激指标及抑郁评分的影响

摘要 目的：探究艾司氯胺酮联合舒芬太尼对剖宫产围手术期镇痛、应激指标及抑郁评分的影响。方法：选取2021年1月~2021年10月期间我院收治的行剖宫产手术的180例产妇,采用随机数字表法分为A组、B组、C组各60例。A组胎儿娩出后即刻静脉输注艾司氯胺酮,术后艾司氯胺酮联合舒芬太尼静脉自控镇痛;B组和C组胎儿娩出后不给予艾司氯胺酮,用生理盐水代替;B组术后静脉自控镇痛与A组相同,C组术后给予舒芬太尼静脉自控镇痛。比较三组产妇术后4 h（T₁）、8 h（T₂）、12 h（T₃）、24 h（T₄）、48 h（T₅）疼痛程度[简化McGill疼痛问卷（SF-MPQ）评分]、镇静程度（Ramsay镇静评分）,记录三组术前1 d和术后1 d的应激指标[去甲肾上腺素（NE）、皮质醇（Cor）、促肾上腺皮质激素（ACTH）]水平,比较三组术前1 d和术后3、7 d抑郁程度[爱丁堡产后抑郁量表（EPDS）],比较三组不良反应发生情况。结果：T₁、T₂、T₃时A组视觉模拟评分（VAS）明显低于B、C组（P均<0.05）,T₁、T₂时A组痛分级指数（PRI）感觉分明显低于B、C组（P均<0.05）,T₁、T₂、T₃、T₄、T₅时A组PRI情感分明显低于B、C组（P均<0.05）,T₁时A组现时疼痛强度（PPI）评分明显低于B、C组（P均<0.05）。T₁、T₂、T₃、T₄、T₅时,三组Ramsay镇静评分比较差异均无统计学意义（P均>0.05）。术后1 d,三组NE、Cor、ACTH水平均较术前1 d显著升高（P均<0.05）,术前1 d和术后1 d,三组NE、Cor、ACTH水平比较差异均无统计学意义（P均>0.05）;术后3、7 d时A组EPDS评分明显低于术前1 d和同时间B、C组（P均<0.05）;三组不良反应发生情况对比无显著性差异（P均>0.05）。结论：艾司氯胺酮联合舒芬太尼在剖宫产围术期镇痛中应用效果良好,能够减轻产妇术后早期抑郁程度。     

Studies on Non-Covalent Interaction of Coumarin Attached Pyrimidine and 1-Methyl Indole 1,2,3 Triazole Analogues with Intermolecular Telomeric G-Quadruplex DNA Using ESI-MS and Spectroscopy

In the present study, electrospray ionization mass spectrometry (ESI-MS) and spectroscopy have been used to evaluate the non-covalent interaction, stoichiometry, and selectivity of two synthetic coumarin-attached nucleoside and non-nucleoside 1,2,3-triazoles, namely, (1-(5-(hydroxymethyl)-4-(4-((2-oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-furan-2-yl)5-methyl pyrimidine-2,4(1H,3H)-dione (Tr1) and 4-((1-((-1-methyl-1H-indol-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (Tr2) with two different human telomeric intermolecular G-quadruplex DNA structures formed by d(T₂AG₃) and d(T₂AG₃)₂ sequences. ESI-MS studies indicate that Tr1 specifically interacts with four-stranded intermolecular parallel quadruplex complex, whereas Tr2 interacts with two hairpin as well as four-stranded intermolecular parallel quadruplex complexes. UV–Visible spectroscopic studies suggest that Tr1 and Tr2 interact with G-quadruplex structure and unwind them. Job plots show that stoichiometry of ligand:quadruplex DNA is 1:1. Circular dichroism (CD) studies of G-quadruplex DNA and Tr1/Tr2 ligands manifest that they unfold DNA on interaction. Fluorescence studies demonstrate that ligand molecules intercalate between the two stacks of quadruplex DNA and non-radiative energy transfer occurs between the excited ligand molecules (donor) and quadruplex DNA (acceptor), resulting in enhancement of fluorescence emission intensity. Thus, these studies suggest that nucleoside and non-nucleoside ligands efficiently interact with d(T₂AG₃) and d(T₂AG₃)₂ G-quadruplex DNA but the interaction is not alike with all kinds of quadruplex DNA, this is probably due to the variation in the pharmacophores and structure of the ligand molecules.     

Diagnostic Confusion Attributable to Spurious Elevation of Both Total Thyroid Hormone and Thyroid Hormone Uptake Measurements in the Setting of Autoantibodies: Case Report and Review of Related Literature

ObjectiveTo review the effect of thyroid autoantibodies on thyroid function assays and to present a case in which thyroid autoantibodies resulted in spurious assay readings for both total thyroid hormone levels and thyroid hormone uptake measurements.MethodsWe present a detailed case, including serial laboratory data, and review the relevant literature.ResultsA 61-year-old man with a history of autoimmune disease presented for evaluation of abnormal results of thyroid function tests. The patient had been treated for hypothyroidism with levothyroxine and was noted to have an elevated total thyroxine (T₄) level in the setting of a low total triiodothyronine (T₃) value and a mildly elevated thyrotropin concentration. He had been referred for evaluation of a presumed deiodinase deficiency that impaired conversion of T₄ to T₃. During treatment with levothyroxine, these test results were confirmed, and the patient was also found to have an elevated T₄ uptake. These findings were initially thought to be due to an excess of transthyretin; however, more extensive testing revealed that the patient had an autoantibody to T₄ that interfered with the assays for both T₄ and T₄ uptake.ConclusionAutoantibodies to both T₃ and T₄ have been described. Such antibodies are not uncommon in patients with thyroid disease. On rare occasions, these antibodies may cause spurious assay readings and obscure the diagnosis. To our knowledge, this is the first report in which both the total T₄ level and the T₄ uptake were elevated because of the presence of autoantibodies. Thyroid hormone autoantibodies must be considered when clinicians encounter patients with unexplained abnormal results of thyroid function tests. (Endocr Pract. 2008; 14:738-742)     

Clamping down on weak terminal base pairs: oligonucleotides with molecular caps as fidelity-enhancing elements at the 5'- and 3'-terminal residues

The base-pairing fidelity of oligonucleotides depends on the identity of the nucleobases involved and the position of matched or mismatched base pairs in the duplex. Nucleobases forming weak base pairs, as well as a terminal position favor mispairing. We have searched for 5′-appended acylamido caps that enhance the stability and base-pairing fidelity of oligonucleotides with a 5′-terminal 2′-deoxyadenosine residue using combinatorial synthesis and MALDI-monitored nuclease selections. This provided the residue of 4-(pyren-1-yl)butyric acid as a lead. Lead optimization gave (S)-N-(pyren-1-ylmethyl)pyrrolidine-3-phosphate as a cap that increases duplex stability and base-pairing fidelity. For the duplex of 5′-AGGTTGAC-3′ with its fully complementary target, this cap gives an increase in the UV melting point T_m of +10.9°C. The T_m is 6.3–8.3°C lower when a mismatched nucleobase faces the 5′-terminal dA residue. The optimized cap can be introduced via automated DNA synthesis. It was combined with an anthraquinone carboxylic acid residue as a cap for the 3′-terminal residue. A doubly capped dodecamer thus prepared gives a melting point decrease for double-terminal mismatches that is 5.7–5.9°C greater than that for the unmodified control duplex.     

丙泊酚联合瑞芬太尼麻醉对直肠癌根治术患者血流动力学和T淋巴细胞亚群的影响

目的:探讨直肠癌根治术患者采用丙泊酚联合瑞芬太尼麻醉维持的临床效果。方法:选取2016年3月～2019年3月期间在我院行直肠癌根治术的患者82例,根据乱数表法将患者分为对照组(n=41)和研究组(n=41),其中对照组给予七氟醚麻醉维持,研究组给予丙泊酚联合瑞芬太尼麻醉维持,比较两组患者围术期指标、血流动力学和T淋巴细胞亚群变化,记录两组围术期不良反应情况。结果:研究组患者的清醒时间、拔管时间均短于对照组(P0.05)。两组患者麻醉成功后(T2)～术后72 h(T5)时间点心率(HR)、平均动脉压(MAP)均呈先降低后升高趋势(P0.05);研究组T2～术后24 h(T4)时间点HR、MAP高于对照组(P0.05)。两组不良反应发生率对比无差异(P0.05)。两组患者T2～T5时间点CD4~+、CD4~+/CD8~+均呈先降低后升高趋势(P0.05),CD8~+呈先升高后降低趋势(P0.05);研究组T_2～T_4时间点CD4~+、CD4~+/CD8~+高于对照组(P0.05),CD8~+低于对照组(P0.05)。结论:直肠癌根治术患者麻醉维持选用丙泊酚联合瑞芬太尼可减少对机体血流动力学的影响,减轻免疫抑制,改善围术期相关指标,且不增加不良反应发生率。