Gender-based differences in COVID-19.

The coronavirus disease (COVID-19) is a novel emerging infectious disease spreading worldwide. To further understand the disease, we compared its clinical characteristics, symptoms and outcomes by gender. In an analysis of public surveillance data of Taiwan from January 21 to April 18, 2020, a total of 398 patients were diagnosed with COVID-19 by the detection of severe acute respiratory syndrome coronavirus 2 in pharynx swabs. We divided the patients into two groups: men and women. The associated data were collected, and multivariate comparisons of radiographic infiltration were conducted to analyse the gender-based differences. The mean incubation period was 5.4 ± 5 days, and the incubation period in men was 3.2 days longer than that in women (8 ± 8.1 vs. 4.8 ± 3, p = 0.05). The male patients with COVID-19 with infiltration in chest X-rays (CXR) were 12 years older than their female counterparts. The mortality rate in the male patients with COVID-19 was 6.4-fold higher than that in the female patients (3.2% vs. 0.5%, p < 0.05). The patients with comorbidities of diabetes mellitus and hypertension were vulnerable to infiltration in CXR and the patients with COVID-19 who had infiltration in CXR easily ended up with intubation, intensive care unit admission and mortality. Moreover, female patients with COVID-19 who had fever, cough and dyspnoea were susceptible to infiltration in CXR. Irrespective of whether the cases were imported female from Europe, America or Asia, indigenous male, the factors associated with death in patients with severe COVID-19 were male sex, elderly, female with fever, cough, dyspnoea and DM.

Sensitivity of mango anthracnose pathogen, Colletotrichum gloeosporioides, to the fungicide prochloraz in Taiwan.

In order to monitor the sensitivity of the mango anthracnose fungus, Colletotricthum gloeosporioides, to the eradicative imidazole fungicide prochloraz, a total of 43 mango orchards were surveyed throughout the Tainan area, covering a 4000 ha region of mango plantations. These orchards were recognized as having undergone higher prochloraz application. A subpopulation, 55 isolates in total, collected from Wufeng, Taichung, served as a baseline population since no fungicide was ever used in this mango plantation. A total of 545 isolates were surveyed, and it was found that the IC50s (50% inhibitory concentration) fell within a range of 0.009-0.1554 microg/ml. No significant resistance was found in the field even with higher frequency of prochloraz application. One orchard (Yujing (Wu)) located in the Yujing area known had a higher frequency of prochloraz application, the IC50s were between 0.0204 - 0.1554 microg/ml. The average IC50 was 0. 0766 microg/ml, which was about five times higher than the baseline population (0.015 microg/ml). A significant t test indicated that these two sub-populations were different at p = 0.01. The results indicated that the dose-response of C. gloeosporioides to prochloraz shifted slightly toward higher IC50 over time. A further survey using 10 microg/ml as the threshold dosage was conducted, and the results of 1375 isolates collected throughout this region showed that no isolate could survive at this dosage. Knowing that the registered dosage for field use is 83.3 microg/ml, based on above results, the author concluded that there was no sign of prochloraz resistance in mango plantations 13 years after prochloraz registration in Taiwan.

Local distribution of mepivacaine after distal interphalangeal joint injection in horses.

OBJECTIVE: To evaluate the distribution of mepivacaine hydrochloride after distal interphalangeal (DIP) joint injection in horses. DESIGN: Prospective, uncontrolled study. ANIMALS: 10 adult horses. PROCEDURE: 30 minutes before euthanasia, 8 ml of 2% mepivacaine hydrochloride was injected into the dorsal pouch of a forelimb DIP joint. Synovial tissue from the DIP joint and podotrochlear (navicular) bursa and bone tissue from the medullary cavity of the distal sesamoid (navicular) bone were taken from both forelimbs immediately after death. All synovial and bone specimens were analyzed for tissue concentration of mepivacaine by high-performance liquid chromatography. Synovial tissue and bone specimen concentrations from the injected forelimb were compared with corresponding specimens from the noninjected forelimb. All synovial tissue and bone specimen concentrations were compared with an estimated effective tissue concentration of mepivacaine (0.3 microgram/mg) for local anesthesia. RESULTS: Specimen concentrations of mepivacaine from the injected forelimb were significantly greater (P < 0.05) than those in the corresponding tissues of the contralateral noninjected forelimb. All DIP joint and navicular bursa synovial tissue specimens from the injected forelimb had greater than the estimated effective tissue concentration of mepivacaine for local anesthesia. Of the 10 navicular bone specimens from the injected forelimb, 4 were higher and 2 were within 20% of the estimated effective tissue concentration of mepivacaine for local anesthesia. CONCLUSIONS: Mepivacaine hydrochloride deposited into the DIP joint should anesthetize pain arising from navicular bursa synovia and may decrease pain arising from the medullary cavity of the navicular bone. CLINICAL RELEVANCE: DIP joint injection of mepivacaine hydrochloride is not specific for DIP joint pain.

High-performance liquid chromatographic analysis of glycoamines in serum.

This report describes the development of an HPLC-UV method for studies of glycoamines and glycoamine-like compounds in normal human serum and osteosarcoma patients serum as potential biological markers of cancer. The glycoamines, a newly recognized class of endogenous, low-molecular-mass biopolymers, are conjugates of amino acids and sugar units, containing 5 to 29 amino acid and 1 to 17 sugar units. After ultrafiltration of serum samples, reversed-phase HPLC separation with diode-array detection was used to obtain standard profiles of serum ultrafiltrates below M(r) 10,000 in healthy subjects. These highly reproducible profiles utilized two-dimensional peak identification and were used to develop a statistical profile of the major glycoamine peaks in normal serum. This newly developed analytical method was subsequently used to address a key question: whether or not there is a single tumor-specific glycoamine or a family of tumor-specific glycoamines in cancer patient serum. Preliminary results suggest that this method can separate and detect glycoamines and glycoamine-like compounds in various types of cancer patients serum with a high degree of reproducibility on the basis of comparative two-dimensional identification of natural compounds and a panel of synthetic glycoamine analogs. Moreover, the method is useful for following the relative changes in the amount of a given glycoamine over an extended clinical time course. Initial results suggest that a glycoamine or glycoamine-like compound, GA-4.63, may have clinical utility in human osteosarcoma studies.

Undiscriminating codon reading with adenosine in the wobble position.

To investigate the reading properties of adenosine in the wobble position we have used site-directed mutagenesis of the Escherichia coli glycine tRNA1(CCC) gene to substitute the nucleotide A in the wobble position of the corresponding tRNA. The effect of this change on the ability of the tRNA to discriminate between the nucleotides in the third position of the glycine codons has been investigated. We have compared the ability of the mutant glycine tRNA1(UCC) and glycine tRNA1(ACC) as well as the mycoplasma glycine tRNA(UCC) to read the glycine codons. The results showed that glycine tRNA1(ACC) unlike glycine tRNA1(UCC) did not fully discriminate between the glycine codons. These experiments were carried out using a new in vitro protein synthesizing system that allows us to monitor the reading of all four glycine codons. In the present paper we give a detailed description of this new in vitro system.

Large-scale methylation patterns in the nuclear genomes of plants.

Methylation was investigated in compositional fractions of nuclear DNA preparations (50-100 kb in size) from five plants (onion, maize, rye, pea and tobacco), and was found to increase from GC-poor to GC-rich fractions. This methylation gradient showed different patterns in different plants and appears, therefore, to represent a novel, characteristic genome feature which concerns the noncoding, intergenic sequences that make up the bulk of the plant genomes investigated and mainly consist of repetitive sequences. The structural and functional implications of these results are discussed.

Reference intervals for eight modified nucleosides in serum in a healthy population from Italy and the United States.

We used a recently devised HPLC method to quantify eight modified nucleosides, an emerging group of tumor markers, in human serum and then calculated their reference intervals in a healthy population from Italy and the United States. We used the statistical procedure of element analysis, which reveals the effects of chosen variables (in this case, nationality, sex, and age) on an analyte (here, modified nucleosides). Using element analysis, we calculated the exact weight of each variable on the reference values. We found that nationality has the greatest effect on the serum concentrations of all the modified nucleosides apart from pseudouridine, whereas sex significantly influences only the concentrations of 4-pyridone-3-carboxamide-N1-ribofuranoside, 1-methylinosine and N2,N2-dimethylguanosine; age affects only N2,N2-dimethylguanosine. Thus, the reference intervals of all the nucleosides except pseudouridine were calculated separately for Italians and Americans, and the reference values for 4-pyridone-3-carboxamide-N1-ribofuranoside, 1-methylinosine, and N2,N2-dimethylguanosine were calculated separately for men and women. Our data form the baseline for study of variations in serum concentrations of modified nucleosides in various pathophysiological conditions.

Identification and structural characterization of O-beta-ribosyl-(1"----2')-adenosine-5"-phosphate in yeast methionine initiator tRNA.

We report in this paper on the complete structure determination of the modified nucleotide A*, now called Ar(p), that was previously identified in yeast methionine initiator tRNA as an isomeric form of O-ribosyl-adenosine bearing an additional phosphoryl-monoester group on its ribose2 moiety. By using the chemical procedure of periodate oxidation and subsequent beta-elimination with cyclohexylamine on mono- and dinucleotides containing Ar(p), we characterized the location of the phosphate group on the C-5" of the ribose2 moiety, and the linkage between the two riboses as a (1"----2')-glycosidic bond. Since the structural difference between phosphatase treated Ar(p) and authentic O-alpha-ribosyl-(1"----2')-adenosine from poly(ADP-Ribose) was previously assigned to an isomeric difference in the ribose2-ribose1 linkage, the (1"----2')-glycosidic bond of Ar(p) was deduced to have a beta-spatial configuration. Thus, final chemical structure for Ar(p) at the position 64 in yeast initiator tRNA(Met) has been established as O-beta-ribosyl-(1"----2')-adenosine-5"-phosphate. This nucleotide is linked by a 3',5'-phosphodiester bond to G at the position 65.

Eukaryotic tRNAs(Pro): primary structure of the anticodon loop; presence of 5-carbamoylmethyluridine or inosine as the first nucleoside of the anticodon.

The modified nucleoside U*, located in the first position of the anticodon of yeast, chicken liver and bovine liver tRNA(Pro) (anticodon U*GG), has been determined by means of TLC, HPLC, ultraviolet spectrum and gas chromatography-mass spectrometry. The structure was established as 5-carbamoylmethyluridine (ncm5U). In addition, we report on the primary structures of the above-mentioned tRNAs as well as those which have the IGG anticodon. In yeast, the two tRNA(Pro) (anticodons U*GG and IGG) differ by eight nucleotides, whereas in chicken and in bovine liver, both anticodons are carried by the same 'body tRNA' with one posttranscriptional exception at position 32, where pseudouridine is associated with ncm5U (position 34) in tRNA(Pro) (U*GG) and 2'-O-methylpseudouridine is associated with inosine (position 34) in tRNA(Pro) (IGG).

Codon discrimination and anticodon structural context.

Site-directed mutagenesis has been used to change the nucleotide C in the wobble position of tRNA(1Gly) (CCC) to U. The mutated tRNA was tested for its ability to read glycine codons in an in vitro protein-synthesizing system programmed with the phage message MS2-RNA that had been modified by site-directed mutagenesis so as to make it possible to monitor conveniently the reading of all four glycine codons. The results showed that while the efficiency of tRNA(1Gly) (UCC) was comparable to that of mycoplasma tRNA(Gly) (UCC) in the reading of the codon GGA, the mycoplasma tRNA(Gly) was far more efficient than the tRNA(1Gly) (UCC) in the reading of the codons GGU and GGC. Thus, the anticodon UCC, when present in the structural context of the tRNA(1Gly) molecule, behaved as predicted by the wobble rules while in the structural context of the mycoplasma tRNA(Gly) it read without discrimination between the nucleotides in the third codon position, in violation of the wobble restrictions. The result with the codon GGG showed that the anticodon UCC, when present in tRNA(1Gly), was considerably less efficient in reading this codon than it was in the structural context of the mycoplasma tRNA(Gly). It would therefore seem that the anticodon UCC, when present in a certain tRNA, can be an efficient wobbler, while in the molecular environment of another tRNA it is markedly restricted in its ability to wobble.

Ribonucleoside analysis by reversed-phase high-performance liquid chromatography.

Over the past fifteen years we have developed and refined the analytical chromatographic methodologies using reversed-phase high-performance liquid chromatography and UV-photodiode array detection (RPLC-UV) for the detection and measurement of the major and modified nucleosides in nucleic acids and biological fluids. RPLC-UV nucleoside analysis as it has now evolved is a powerful new research tool to aid investigators in the fields of biochemical and biomedical research. This RPLC-UV nucleoside method can resolve more than 65 nucleosides in a single analysis with "run-to-run" peak retention variations of less than 1%. A complete nucleoside composition can be obtained from as little as 0.5 micrograms RNA. Identification and confirmation of nucleosides can be made from the highly reproducible retention times and from the characteristic UV spectrum from a few picomoles (ca. 1 ng) of nucleoside. In this paper we introduce standard RPLC-UV methodologies for the analysis of nucleosides and nucleoside composition of RNAs. The chromatographic protocols and standard nucleoside columns are presented and the essential requirements necessary in the HPLC instrumentation are described. Three optimized RPLC systems were developed, each with particular emphasis placed on resolution, speed, or sensitivity. In addition, three unfractionated tRNAs were selected as sources of reference nucleosides and for assessment of the performance of the chromatography. From these tRNAs, a large array of nucleosides were characterized which are used in standardization and calibration of the method. Also discussed is the use of a diode-array detector for enhancement of the reliability of nucleoside identification and accuracy of measurement. An extended enzymatic hydrolysis protocol for the liberation of exotically modified nucleosides in tRNAs is also described. Chromatographic retention times and UV spectra for a large number of ribonucleosides are tabulated. The RPLC-UV ribonucleoside analytical protocols are capable of quantifying 31 nucleosides. Approximately 1 microgram of an isoaccepting tRNA, or 20 micrograms of unfractionated tRNA are needed for quantitative analysis. With this amount of tRNA, the percent relative error of measurement of the four major nucleosides is less than 2%, and for the modified nucleosides about 5%. As little as 0.2 micrograms of pure isoaccepting tRNA can be analyzed, but at the expense of precision as at this low sample size a 20-30% relative error for modified nucleosides is to be expected. For quantitation of the modified nucleosides in rRNA, which contains much less modification than tRNAs, 10-100 micrograms of sample are needed per injection.(ABSTRACT TRUNCATED AT 400 WORDS)

Classification of lung cancer patients and controls by chromatography of modified nucleosides in serum.

A wide spectrum of modified nucleosides has been quantified by high-performance liquid chromatography in serum of 49 male lung cancer patients, 35 patients with other cancers, and 48 patients hospitalized for nonneoplastic diseases. Data for 29 modified nucleoside peaks were normalized to an internal standard and analyzed by discriminant analysis and stepwise discriminant analysis. A model based on peaks selected by a stepwise discriminant procedure correctly classified 79% of the cancer and 75% of the noncancer subjects. It also demonstrated 84% sensitivity and 79% specificity when comparing lung cancer to noncancer subjects, and 80% sensitivity and 55% specificity in comparing lung cancer to other cancers. The nucleoside peaks having the greatest influence on the models varied dependent on the subgroups compared, confirming the importance of quantifying a wide array of nucleosides. These data support and expand previous studies which reported the utility of measuring modified nucleoside levels in serum and show that precise measurement of an array of 29 modified nucleosides in serum by high-performance liquid chromatography with UV scanning with subsequent data modeling may provide a clinically useful approach to patient classification in diagnosis and subsequent therapeutic monitoring.

Presence of phosphorylated O-ribosyl-adenosine in T-psi-stem of yeast methionine initiator tRNA.

We report in this paper on isolation and characterization of two unknown nucleosides G* and [A*] located in the T-psi-stem of yeast methionine initiator tRNA, using the combined means of HPLC protocols, real time UV-absorption spectrum, and post-run mass spectrometry by electron impact or fast atom bombardment. The G* nucleoside in position 65 was identified as unmodified guanosine. The structure of the unknown [A*] in position 64 was characterized as an isomeric form of O-ribosyl-adenosine by comparison of its chromatographic, UV-spectral and mass spectrometric properties with those of authentic O-alpha-ribofuranosyl-(1"----2')-adenosine isolated from biosynthetic poly(adenosine diphosphate ribose). Our studies also brought evidence for the presence of a phosphorylmonoester group located on this new modified nucleoside [A*], when isolated by ion exchange chromatography from enzymic hydrolysis of yeast initiator tRNAMet without phosphatase treatment.

Reduced genomic 5-methylcytosine content in human colonic neoplasia.

DNA methylation appears to play an important role in both physiological and experimentally modified gene expression, and alterations in DNA methylation have been described in animal tumor models and in transformed cells and tumor cell lines. However, there have been comparatively few reports on DNA methylation in primary human malignancies, and these reports are somewhat contradictory. While individual genes have shown hypomethylation in colon cancer and premalignant adenomas as well as in lung cancer, other genes have shown increased methylation, and absolute measures of 5-methylcytosine content have shown decreases in malignancies but not in premalignant adenomas. We have used a sensitive quantitative measurement of 5-methylcytosine content by high performance liquid chromatography revealing an unequivocal hypomethylation of tumor DNA. An average of 8 and 10% reduction in genomic 5-methylcytosine content was seen in apparently all colon adenomas and adenocarcinomas, respectively, and there was no significant difference between benign and malignant tumors. This is a substantial quantitative alteration and suggests a pervasive abnormality in the control of DNA methylation. Surprisingly, three patients with the highest 5-methylcytosine content in their normal colon appear to have a germline predisposition to cancer (Lynch syndrome).

High-performance liquid chromatographic-radioimmunoassay method for the measurement of angiotensin II peptides in human plasma.

A highly selective high-performance liquid chromatographic-radioimmunoassay method for the measurement of individual endogenous angiotensin peptides in human plasma is described. This method allows the complete resolution of the immunoreactive angiotensin II peptides. We have also measured the angiotensin peptide levels and compared them in both pooled and individual human plasma. The effects of inhibition of angiotensin-converting enzyme on the angiotensin peptide levels have also been observed in a patient with renovascular hypertension with the plasma angiotensin II level being reduced greater than seven-fold. This new methodology was validated by recovery experiments in plasma over a range of physiological levels using two methods of detection, radioimmunoassay and liquid scintillation counting. Consistent recoveries near 80% have been achieved for each peptide in plasma at concentrations over a physiological range. The described method enables the direct measurement of the circulating angiotensin peptides and the elucidation of their specific roles in physiological and disease states.

Amino acid analysis by capillary gas chromatography.

Two developments have enabled major advancements in the use of capillary gas chromatography (GC), the result being its much more widespread use in investigations on a broad range of chemical and biological problems. The 2 technological developments were the introduction of fused silica capillary columns and the development of immobilized stationary phases for capillary GC columns. Because fused silica columns with immobilized stationary phases of varying polarities are offered by numerous vendors of chromatographic equipment, they have become widely used for many analytical tasks. We conducted a study to compare the effectiveness of commercially available fused silica capillary columns with the classical ion-exchange method in the separation and quantitation of amino acids. We selected the N-trifluoroacetyl (TFA) n-butyl and the N-heptafluorobutyryl (HFB) isobutyl ester derivatives for this study because of the extensive research and application of these derivatives during the past 20 years. The amino acid content of hydrolysates of 5 materials was measured: ribonuclease, beta-lactoglobulin, lysozyme, soybean meal, and a commercial poultry feed. Single 6N HCl hydrolysates of each material were prepared to minimize sample preparation differences, and 3 independent analyses of each hydrolysate were made by each of 3 techniques: the N-TFA n-butyl and N-HFB isobutyl ester methods using capillary gas chromatography and the ion-exchange chromatographic method using a Beckman 121 M amino acid analyzer. Our results clearly demonstrate that capillary GC analysis of amino acids using fused silica bonded-phase columns provides data with good precision and in general excellent agreement with ion-exchange analyses.

N4-methylcytosine as a minor base in bacterial DNA.

The DNA base composition, including the minor base content, of 26 strains of bacteria was determined. The studied bacteria are sources of widely used restriction endonucleases. Approximately 35% of the bacterial DNAs contained N4-methylcytosine, about 60% contained 5-methylcytosine, and about 90% had N6-methyladenine.

Analysis of amino acids by gas chromatography as the N-trifluoroacetyl n-butyl esters.

This presentation describes amino acid analysis with the gas chromatographic method and experimental conditions using the N-trifluoroacetyl n-butyl ester derivatives; the study we describe here was undertaken to compare gas chromatographic (GC) and ion-exchange chromatographic (IEC) analyses of amino acids in hydrolysates of 9 diverse sample types to gain insight into effects of these 2 chromatographic methods of analysis on variation in amino acid results. Our study showed that values for samples prepared by 2 separate laboratories using the same procedure were generally in good agreement when all of the hydrolysates were analyzed by a single laboratory using a single method of analysis. To compare results from gas chromatography with those from ion-exchange chromatography analyses were performed by 2 different laboratories on the same hydrolysates and on different hydrolysates prepared by the same method by both laboratories. The data demonstrate that GC and IEC can be expected to yield essentially identical results when applied to the same hydrolysate. Agreement is so close that interlaboratory differences in hydrolysate preparation of the same sample contribute as much to variation in amino acid results as does the method of analysis, a fact which should be noted in planning collaborative studies.

Antisuppressor mutations and sulfur-carrying nucleosides in transfer RNAs of Schizosaccharomyces pombe.

Antisuppressor mutations reduce the efficiency of nonsense suppressors. A mutation in the gene sin4 of Schizosaccharomyces pombe leads to loss of 5-(methoxycarbonylmethyl) thiouridine (mcm5s2U) from the first anticodon position of tRNAs. This resembles the phenotype of sin3 (Heyer, W. D., Thuriaux, P., Kohli, J., Ebert, P., Kersten, H., Gehrke, C., Kuo, K. C., and Agris, P. F. (1984) J. Biol. Chem. 259, 2856-2862), but the mutations reside in different genes. In vivo 35S-labeled tRNA from the parental suppressor strain sup3, the antisuppressor strains sin3 and sin4, and the double mutant sin3 sin4 has been digested to nucleosides and analyzed with high performance liquid chromatography methods. The major sulfur-carrying nucleoside in wild-type S. pombe tRNA is mcm5s2U. It is reduced in the mutant strains. Two other thiolated nucleosides are also present: 2-thiouridine and a nucleoside of unknown structure. Neither was affected by the antisuppressor mutations. Thiocytidine has not been found. Independent from their effect on suppressors, the two mutations sin3 and sin4 reduce the growth rate of cells, and sin3 also increases cell length. In vivo decoding of the serine codon UCG by the UCA reading serine tRNA is not promoted by the two antisuppressor mutations.

Quantitative measurement of mRNA cap 0 and cap 1 structures by high-performance liquid chromatography.

Viral and eukaryotic mRNA molecules have a unique 5'-end. The 5'-terminus consists of m7G(5')ppp(5')N'(m)pN''(m), which is termed a "cap" structure. The study of these cap structures has led to the development of many methods of identification and analysis. Many of the methods have been time-consuming or have not been able to distinguish between the different caps, and they are quantifiable only by employing radiolabels. This paper presents the use of reversed-phase high-performance liquid chromatography as a rapid and efficient tool for the separation, identification and quantitation of caps. An ion-exchange enrichment procedure was also developed for the isolation of cap 0 and cap 1 structures from unfractionated RNAs. The recoveries of different caps ranged from 83 to 99%, with a relative standard deviation range of 1.3-4.4%. In this method, caps were released from commercially obtained rabbit globin mRNA by nuclease P1 digestion. The products of digestion were treated with alkaline phosphatase and separated on an octadecylsilyl column using stepwise or gradient elution. Cap structures and any internal modified nucleosides were identified by their retention times and UV spectra relative to reference compounds. The amount of each cap 0 or cap 1 structure was determined by its UV absorbance relative to a known quantity of reference compound. This method allows the quantitation of 0.2 nmol or more of cap 0 and cap 1 structures. Total UV spectra can be obtained for 0.5 nmol or more of cap. This methodology permits investigations on viral and eukaryotic mRNA cap biosynthesis and turnover during viral transformation, differentiation, cap synthesis in the cell cycle, etc.