Biochemical evaluation of the morphogenetic potential of wheat callus cells in vitro

Morphogenetic processes in immature embryos of wheat (Triticum aestivum L.) were investigated using a molecular marker for meristematic cells of cereals (proliferative antigen of meristem initial cells, PAIC). The used genetic model comprised a series of almost isogenic lines of wheat alternative by the alleles of dwarfing genes (RhtB1c, RhtB1b, and Rht14) and the original cv. Saratovskaya 29. The Rht genetic system was found to affect the level of PAIC in the wheat callus cells during callus formation and subsequent regeneration. We found differences in the level of PAIC at certain stages of culturing and a general trend of its content in the course of callus formation and secondary differentiation in the callus tissue. Possible role of PAIC as a marker of meristematic centers within the callus tissue and the prospect of its use in breeding as an additional criterion for estimation of morphogenetic ability of the newly produced lines of wheat are discussed.     

Molecular systematics and species limits in the Philippine fantails (Aves: Rhipidura)

Islands have long-attracted scientists because of their relatively simple biotas and stark geographic boundaries. However, for many islands and archipelagos, this simplicity may be overstated because of methodological and conceptual limitations when these biotas were described. One archipelago that has received relatively little recent attention is the Philippine islands. Although much of its biota was documented long ago, taxonomic revision and evolutionary study has been surprisingly scarce, and only a few molecular phylogenetic studies are beginning to appear. We present a molecular phylogeny and taxonomic revision for the Philippine fantails (Aves: Rhipidura) using nuclear and mitochondrial DNA sequences. Our results suggest that current taxonomy underestimates diversity in the group. Some morphologically distinct subspecies warrant species status, whereas one was indistinguishable genetically and morphologically and should not be retained. A few taxa require additional sampling for thorough taxonomic assessment. Patterns of diversity within Philippine Rhipidura mostly corroborate predictions of the Pleistocene aggregate island complex (PAIC) hypothesis, in which diversity is expected to be partitioned by deep water channels separating Pleistocene aggregate islands rather than by current islands. Substantial structure within PAIC clades indicates that additional drivers of diversification should be considered.     

Species‐specific responses to island connectivity cycles: refined models for testing phylogeographic concordance across a Mediterranean Pleistocene Aggregate Island Complex

The contribution of Pleistocene sea level changes to diversification patterns in archipelagos around the world, and specifically whether the repeated cycles of island connectivity and isolation acted as a ‘species pump’ is debated. The debate has been perpetuated in part because of the type of evidence used to evaluate the species‐pump hypothesis. Specifically, existing tests of the ‘Pleistocene Aggregate Island Complex’ (PAIC) model of diversification interpret the lack of concordant divergence times among multiple codistributed taxa as a rejection of the PAIC model. However, the null expectation of concordance disregards taxon‐specific ecological traits and geographic characteristics that may affect population persistence and gene flow among islands. Here, we study the factors affecting population divergence in thirteen flightless darkling beetle species (Coleoptera: Tenebrionidae) across the PAIC system of the Cycladic plateau in the Aegean archipelago. Based on isolation‐by‐resistance analyses, hierarchical amova and the degree of genealogical sorting on individual islands, we identify a major effect of bathymetry and habitat stability on the levels of genetic divergence across the PAIC, with island size and body size playing a secondary role as well. We subsequently use bathymetric maps and habitat association to generate predictions about the set of islands and group of taxa expected to show phylogeographic concordance. We test these predictions using hierarchical approximate Bayesian computation and show how our interpretations regarding the role of PAICs as drivers of divergence change when relying on a null expectation of concordance compared to a refined model that takes geography and ecological traits into account.     

Ambrosia elatior (L.) in Hungary (1989–1990)

Summary Weeds and among themAmbrosia are probably the most important vascular plants related to pollinosis in Hungary. Sampling was carried out in central (Budapest) and in southern (Paks, Szeged) Hungary. The results of two years (1989–1990) of aerobiological study onAmbrosia airborne pollen are reported. The highest percentage of airborne pollen was found in the mid-August to mid-September period, having a good correlation with clinical data on pollinosis. The implications of these results are considered in the context of forecasting and prevention of seasonal ragweed pollinosis.     

Northward geographic diversification of a kleptoparasitic spider Argyrodes lanyuensis (Araneae,Theridiidae) from the Philippine Archipelago to Orchid Island

Oceanic islands are unique geographic systems that promote local adaptations and allopatric speciation in many of their highly endemic taxa. This is a common case in the Philippine Archipelago, where numerous unrelated taxa on islands have been inferred to have diversified in isolation. However, few cases have been reported in invertebrates especially among parasitic organisms. Here, we tested for biogeographical structure in novel populations of the “generalist" kleptoparasitic spider, Argyrodes lanyuensis Yoshida, Tso & Severinghaus, 1998 in the Philippines. Results showed that, in addition to Orchid/Lanyu Island, this species has a wide geographic distribution in the Philippine Archipelago. The estimated divergence time of this lineage using the mitochondrial cytochrome oxidase 1 (mt‐CO1) suggests that this species diverged ca 3.12 MYA, during the Pliocene. Two reciprocal monophyletic clades were elucidated in A. lanyuensis, but with limited differentiation across Pleistocene Aggregate Island Complex (PAIC) boundaries and modern‐day islands. However, in our analyses of morphological variation, we identified two phenotypically differentiated units in males (Orchid Island, Taiwan + Luzon, Philippine PAIC populations vs. Palawan + West Visayan + Mindanao PAIC populations). We infer that this species diverged in the southern portion of the Philippine Archipelago and only recently colonized Orchid Island. Our study provides new information on the extensive distribution of A. lanyuensis outside Orchid Island, Taiwan, but we documented a very limited geographically associated genetic variation. Our study points to behavioral phenomena such as foraging behavior as essential contributor to the evolutionary process of species diversification, in contrast to the traditionally invoked geographic drivers of divergence.     

Limitations of tpi and bg genes sub-genotyping for characterization of human Giardia duodenalis isolates

The intestinal protozoan Giardia duodenalis includes 2 genetically distinct assemblages, A and B, which are responsible for human infections. Little is known so far on the genotypes of G. duodenalis human isolates in France. The present characterization of 19 French clinical isolates was aimed at determining their genotype patterns and associations with clinical symptoms, and in vivo metronidazole resistance, respectively. Based on both triose-phosphate isomerase (tpi) and β-giardin (bg) gene sequences, twelve isolates were identified as assemblage A, and 7 as assemblage B for the 2 gene loci. Sub-genotyping heterogeneities were observed in 15/19 isolates attributed to either A or B assemblage. They include frequent mismatches and intra-assemblage discordances and mixed positions, which were found more frequently in tpi than in bg sequences, and in assemblage B than in assemblage A sequences. No association was found between sub-genotypes, clinical symptoms and metronidazole sensitivity. Present data underline the need for improvements in the standardization of G. duodenalis multilocus genotyping approach for further molecular epidemiologic studies of giardiasis.     

Multiple copies of functional, Tet(M)-encoding Tn916-like elements in a clinical Enterococcus faecium isolate

Tn916 and similar elements are very common in clinical enterococcal isolates, and are responsible for transmission of a variety of resistance determinants. It is commonly assumed that clinical strains carrying Tn916 have a single copy, although the actual number of copies in clinical isolates has never been systematically studied. We report a clinical isolate of Enterococcus faecium in which three distinct and excision-proficient copies of Tn916-like elements are present in the genome. All of the elements contain tet(M) genes, at least one of which confers resistance to tetracycline and minocycline. Two elements (Tn6085a, Tn6085b) are indistinguishable, containing an inserted 2758 bp Group II intron at the start of open reading frame Tn916ORF_06. The third (Tn6084) also contains the intron, but also has an ISEfa11 integrated upstream of tet(M). All three copies are able to excise from plasmid vectors when cloned in E. coli, and at least two of the elements can transfer to an E. faecium recipient strain. These data indicate that nearly identical Tn916-like elements encoding Tet(M)-mediated tetracycline/minocycline resistance can coexist in clinical E. faecium isolates.     

Discrepancies between MIC and MLC values of amphotericin B against isolates ofAspergillus species

There is little information addressing the phenomena of discrepancy between minimal inhibitory concentrations (MIC) and minimal lethal concentrations (MLC) values of amphotericin B (AMB) to clinical isolates of fungi. This study assessed in vitro activity of AMB against 70 clinical isolates of aspergilli: 30 strains ofAspergillus fumigatus, 20 strains ofAspergillus flavus and 20 strains ofAspergillus niger. Susceptibility tests were accomplished using a macro broth dilution procedure, with special emphasis on ascertainment of MLCs. AMB exhibited low MIC values against all clinical isolates. While we did not identify any AMB resistant isolates among 70Aspergillus spp. studied as judged by MIC levels, analysis of the data demonstrated a clear discrepancy between the MIC and MLC levels of AMB obtained against clinical isolates ofAspergillus spp. The MLC values of AMB were significantly higher than the MIC values with MIC 50 and MIC 90 of 0.29 and 0.5 µg/ml, respectively, at the second reading time, and MLC 50 and MLC 90 of 2.31 and 9.24 µg/ml, respectively (p<0.001). Additionally, minimal lethal concentrations in 36/70 (51%) of aspergillal isolates studied produced drug concentrations above those which can usually be sustained in patient plasma or tissue.     

Complete sequence of p53 gene in 20 patients with lung cancer: comparison with chemosensitivity and immunohistochemistry

In this study the entirep53 complementary DNA has been sequenced in 20 non-small cell lung carcinomas (NSCLC) and the results correlated with chemosensitivity, immunohistochemistry and clinical data. Ten patients had mutations inp53, 8 missense mutations and 2 nonsense mutations. The method discovered two mutations never described previously and two other mutations that have never been described before in connection with NSCLC tumours. Chemosensitivity data, according to a short-term assay (FMCA), indicated that tumours with p53 mutation were more resistant to cisplatin and cyclophosphamide. Immunohistochemical studied demonstrated a 70% concordance between over-expression of p53 protein and mutation inp53. No conclusions or trends could be drawn from the immunohistochemical studies ofBcl-2 andBax.     

Molecular subtyping of clinical isolates of <Emphasis Type="Italic">Candida albicans</Emphasis> and identification of <Emphasis Type="Italic">Candida dubliniensis</Emphasis>in Malaysia

The genotypes of 221 recent isolates of Candida albicans from various clinical specimens of 213 patients admitted to the University Malaya Medical Centre, Malaysia was determined based on the amplification of a transposable intron region in the 25 S rRNA gene. The analyses of 178 C. albicansisolated from nonsterile clinical specimens showed that they could be classified into three genotypes: genotype A (138 isolates), genotype B (38 isolates) and genotype C (2 isolates). The genotyping of 43 clinical isolates from sterile specimens showed that they belonged to genotype A (29 isolates), genotype B (10 isolates), genotype C (2 isolates) and genotype D (2 isolates). The overall distribution of C. albicans genotypes in sterile and nonsterile specimens appeared similar, with genotype A being the most predominant type. This study reported the identification of C. dubliniensis (genotype D) in 2 HIV-negative patients with systemic candidiasis, which were missed by the routine mycological procedure. The study demonstrated the genetic diversity of clinical isolates of C. albicans in Malaysia.     

Epidemiology of the dermatophytoses in the Florence area of Italy: 1985–1990

Between 1985 and 1990 we observed 2085 cases of dermatophytoses in the Florence area of Italy.Trichophyton rubrum andMicrosporum canis were the major etiological agents. 126 cases of dermatophytoses observed during this period were caused byTrichophyton mentagrophytes. Most of the patients came from rural areas or they kept pets.Epidermophyton floccosum ranked fourth in frequency (95 cases). The patients were adults and mainly males, most of whom engaged in sports or lived in group environments.Microsporum gypseum was isolated in 31 cases. Infections occurred directly from contact with soil or domestic animals. We report the principal clinical data concerning these last three dermatophyte infections.     

马钱子致大鼠体内毒性的研究*——“装袋”算法和16S rRNA基因测序技术在毒理学研究中的应用

目的 中药马钱子（Strychnos nux-vomica L.,SN）在临床上具有消肿止痛的功效,然而,由于含有生物碱类成分,马钱子具有一定毒性。人们对马钱子毒性所引起的大鼠内源性代谢变化及其对肠道微生物群代谢失调的潜在影响知之甚少,因此,马钱子的毒理学研究对其安全性评价具有重要意义。本研究将代谢组学和16S rRNA基因测序技术相结合来探索马钱子的致毒机制。方法 通过急性、蓄积性和亚急性毒性试验,分别确定马钱子的中毒剂量、毒性强度和毒性靶器官。超高效液相色谱-质谱联用技术用于分析大鼠灌胃马钱子后的血清、肝脏和肾脏样本。利用基于装袋算法的决策树和K最近邻（K nearest neighbor,KNN）模型对组学数据进行分类。从大鼠粪便中提取样本后,使用高通量测序平台对细菌的16s rRNA V3-V4区域进行分析。结果 装袋算法提高了样本分类的准确率。共鉴定出12个生物标志物,这些生物标志物的代谢失调可能是马钱子致体内毒性的原因。拟杆菌、粪厌氧棒菌、颤螺菌、双茎体菌等与肾肝功能的生理指标密切相关,这表明马钱子引起的肝肾损害可能与这些肠道细菌的代谢紊乱有关。结论 本文揭示了马钱子的体内致毒机制,为马钱子临床上的安全合理使用提供了科学依据。     

Detection of Malaysian methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> (MRSA) clinical isolates using simplex and duplex real-time PCR

The aim of this study was to develop a methicillin-resistant Staphylococcus aureus (MRSA) detection method based on the melting temperature analysis profiling of S. aureus clinical isolates from three different hospitals in Malaysia. Simplex and duplex real-time PCR assay was used for the simultaneous detection of nuc (species-specific) and mecA (methicillin-resistance) genes in a single SYBR Green I real-time PCR tube assay. Evaluations were based on the melting temperature (T _m) analysis of the amplicons using 23 S. aureus clinical isolates including three ATCC S. aureus standard strains. Real-time PCR amplification products with melting peaks at 78.39 ± 0.4°C and 74.41 ± 0.6°C were detected for nuc and mecA genes, respectively. Each real-time PCR assay was completed within two hours. This rapid genotypic method is useful for the detection of resistant determinant (mecA) and identification of S. aureus (nuc) clinical isolates, thus benefiting patient therapy in hospitals.     

The RHD variants in Chinese population

The Rhesus (Rh) blood group system is the most important blood group system in hemolytic disease of the fetus and newborn (HDFN). In clinical transfusions, the D antigen in the Rh blood group system comes third, behind antigens A and B which from ABO blood group system. Over the past decade, molecular technologies have been used to investigate the RHD allele in different ethnic groups. This review first introduces the basic structure of RhD protein and coding genes, then focuses on D-negative, weak D, partial D, DEL, RhD_null variants reported in the Chinese population. To date, more than 460 RHD variants have been reported around the world, but less than 70 RHD variants have been reported in the Chinese population. Further research is needed to identify more RHD polymorphism and establish criteria for blood detection and transfusion guidelines for RHD variants. Only in this way can we better guarantee the safety of blood transfusion and prevent the occurrence of HDFN. With the accumulation of research and clinical data, we should be clearer which RHD variants are to be regarded as RhD negative and which need to be regarded as RhD positive.     

Effects of acorn masting on population dynamics of three forest-dwelling rodent species in Hokkaido,Japan

The effects of the abundance of acorns of the oak, Quercus crispula, on the population dynamics of three rodent species (Apodemus speciosus, A. argenteus, and Clethrionomys rufocanus) were analyzed using time series data (1992–2006). The data were obtained in a forest in northern Hokkaido, Japan, by live trapping rodents and directly counting acorns on the ground. Apodemus speciosus generally increased in abundance following acorn masting. However, the clear effect of acorn abundance was not detected for the other two rodent species. Acorns of Q. crispula contain tannins, which potentially have detrimental effects on herbivores. Apodemus speciosus may reduce the damage caused by acorn tannins with tannin-binding salivary proteins and tannase-producing bacteria, whereas such physiological tolerance to tannins is not known in the other two rodent species. The differences in the effects of acorns between the three species may be due to differences in their physiological tolerance to tannins.     

Detection of streptomycin resistance in Mycobacterium tuberculosis clinical isolates from China as determined by denaturing HPLC analysis and DNA sequencing

China is regarded by the World Health Organization as a major hot-spot region for Mycobacterium tuberculosis infection. Streptomycin has been deployed in China for over 50 years and is still widely used for tuberculosis treatment. We have developed a denaturing HPLC (DHPLC) method for detecting various gene mutations conferring drug resistance in M. tuberculosis. The present study focused on rpsL and rrs mutation analysis. Two hundred and fifteen M. tuberculosis clinical isolates (115 proved to be streptomycin-resistant and 100 susceptible by a routine proportional method) from China were tested to determine the streptomycin minimal inhibitory concentration (MIC), and subjected to DHPLC and concurrent DNA sequencing to determine rpsL and rrs mutations. The results showed that 85.2% (98/115) of streptomycin-resistant isolates harbored rpsL or rrs mutation, while rpsL mutation (76.5%, 88/115) dominated. MIC of 98 mutated isolates revealed no close correlation between mutation types and levels of streptomycin resistance. No mutation was found in any of the susceptible isolates. The DHPLC results were completely consistent with those of sequencing. The DHPLC method devised in this study can be regarded as a useful and powerful tool for detection of streptomycin resistance. This is the first report to describe DHPLC analysis of mutations in the rpsL and rrs genes of M. tuberculosis in a large number of clinical isolates.     

研究报告: TMEM163变异致髓鞘形成低下患者随访两例及iPSC构建

目的 探讨TMEM163变异导致髓鞘形成低下性脑白质营养不良（HLD）患者的临床特征及遗传学特点，归纳自然病程，以提高对该疾病认识，并构建患者来源诱导多能干细胞，为致病机制研究建立基础。方法 随访2009~2022年北京大学第一医院儿科就诊的2例TMEM163变异致病患儿，对临床表现、遗传学数据、蛋白质结构数据进行分析，总结临床遗传学特点，并采集患儿外周血构建诱导多能干细胞。结果 临床特点：2例患儿均具有早期运动语言发育迟缓、头颅磁共振成像显示脑白质髓鞘化不良，且症状随生长发育逐渐减轻的特点；均于婴儿期起病，以眼球震颤为首发症状，学龄期症状好转。遗传学特点：2例患儿均为TMEM163同一位点新发错义变异c.227T>G p.（L76R）、c.227T>C p.（L76P），均为国际上首例报道。病例2来源外周血单个核细胞成功重编程为诱导多能干细胞。结论 本研究为国际首次随访TMEM163变异致病髓鞘形成低下性脑白质营养不良患儿，完善了其自然病程，扩展了对髓鞘形成低下性脑白质营养不良临床表型认识，并首次构建了TMEM163 c.227T>C p.（L76P）变异患者来源诱导多能干细胞，为进一步致病机制研究打下基础。     

Clinical significance of allergic sensitization toOlea europaea L. pollen in Naples area,Italy

Summary The aim of this study is the evaluation of clinical significance of the allergic sensitization toOlea europaea L. pollen allergens in the Naples area, because in the last 10 years an increasing frequency of IgE mediated sensitization to these allergens has been observed. 4124 patients of both sexes (2989 adults and 1153 children) consecutively examined in our Allergological Center from January 1, 1988 to December 30, 1989 have been considered in this study. The patients were selected on the basis of a history of rhinitis (R) and/or asthma (A) and were evaluated by clinical examination, SPT, RAST and specific or aspecific BPT. Among 2267 adults and 879 children sensitized to almost one aeroallergen we found respectively 306 (13.49%) and 75 (8.53%) subjects with an allergic sensitization toOlea europaea L. pollen. The frequency of clinical manifestation was respectively R+A (58.16%), R (39.54%), A (2.28%) in adults; R+A (61.33%), R (26.66%), A (12%) in children. A monosensitization toOlea europaea L. pollen was found in 1.33% of children and in 2.28% of adults examined. The association with other allergens especially with mites and/or other pollen was very common.     

A neonatal polyvisceral failure linked to a de novo homoplasmic mutation in the mitochondrially encoded cytochrome b gene

Mutations within the mitochondrially encoded cytochrome b (MTCYB) gene are heteroplasmic and lead to severe exercise intolerance. We describe an unusual clinical presentation secondary to a novel homoplasmic mutation within MTCYB. The m.15635T > C transition (S297P) was carried by a newborn who presented with a polyvisceral failure. This mutation was responsible for a complex III deficiency. It was homoplasmic in all tissues tested and was undetectable in patient’s mother. Functional analyses, including studies on patient’s cybrid cell lines, demonstrate the pathogenicity of this variant. Our data show that mutations within MTCYB can be responsible for severe phenotype at birth.     

Development of survivorship model for UV-B irradiated Catla catla larvae

A survivorship model was developed for UV-B irradiated Catla catla (17 days) larvae with the help of Kaplan and Meier Product-Limit (PL) method. Larvae were exposed to UV-B radiation (145 μW cm⁻²) for three different exposure times: 5, 10 and 15 min on every other day. The mean survival time of fish was calculated for each treatment using uncensored and censored survival data during 74 days study period. The mean uncensored and censored survival data for the 5-min exposed fish were 7 and 43, respectively. In 10-min exposure period, the uncensored and censored survival data were 19 and 31, respectively. During maximum exposure of 15 min, the uncensored survival data was 20 and censored data was 30. The mean survival time of fish calculated using PL estimate in 5, 10 and 15-min exposure treatments were 69.61 ± 0.50, 65.25 ± 0.96 and 60.60 ± 1.55 days, respectively. The mean survival time showed a decreasing trend with the increase of exposure period. The survival time was significantly (P < 0.001) higher in 5-min exposure treatment than others. This is clear from the present study that the exposure of UV-B radiation affects the survival rate of surface feeder catla larvae.