浙江省副溶血性弧菌03：K6血清型菌株多位点序列分型研究

目的掌握浙江省不同来源的副溶血性弧菌O3：K6血清型菌株的分子分型特征,为副溶血性弧菌食源性疾病的预防控制提供技术支持。方法选择副溶血性弧菌的7个管家基因dnaE、gyrB、recA、dtdS、pntA、pyrC及tnaA,对62株不同来源的副溶血性弧菌03：K6血清型菌株样本进行PCR扩增、测序,Chromas软件和DNAStar软件分析,核酸序列上传至MLST数据库进行比对,获得每株菌的序列型,绘制多位点序列分型遗传进化树并进行亲缘性分析。结果62株副溶血性弧菌03：K6血清型菌株中,59株菌为ST-3型（3,4,19,4,29,4,22）,1株菌为ST-121型（3,2,82,52,4,78,66）,1株菌为新的ST型（5,10,34,27,77,49,23）,1株菌仅gyrB基因第562位点由C突变为T,其余基因序列与ST4型（3,5,22,12,20,22,25）一致。结论浙江省副溶血性弧菌03：K6血清型菌株的主要MLST型别为ST-3型。     

杭州地区单核细胞增生李斯特菌食品分离株分子型别研究

目的研究杭州地区单核细胞增生李斯特菌(Listeria monocytogenes)食品分离株的分子分型情况,了解当地流行株的型别特征。方法用脉冲场凝胶电泳(PFGE)和多位点序列分型(MLST)的方法对单核细胞增生李斯特菌进行分子分型。PFGE结果进行聚类分析,并绘制MLST数据的最小生成树。结果 6个血清型组成的133株杭州食品分离株共获得19个MLST型别,并发现1个新的ST型ST767。ST9和ST121是数量最多的型别。用AscI和ApaI酶切分别获得33和45个PFGE带型。结论杭州地区单核细胞增生李斯特菌食品分离株分子型别分布广泛,大部分菌株是可引起人李斯特菌病的Lineage I和Lineage II菌株。食品中单核细胞增生李斯特菌的污染比较严重,应加强监测与管理以防食源性疾病的发生。     

碳青霉烯类抗生素耐药肺炎克雷伯菌耐药基因分布和分子分型

目的 探讨碳青霉烯类耐药肺炎克雷伯菌的耐药基因分布及分子分型特征。方法 收集38株临床分离的肺炎克雷伯菌,采用肉汤稀释法药敏实验、改良Hodge试验、PCR电泳、脉冲场凝胶电泳分型及多位点序列分型测定其耐药性、耐药基因型及分子型别。结果 对碳青霉烯类药物亚胺培南、美罗培南、厄他培南耐药率分别为18.42%(7/38)、28.95%(11/38)、34.21%(13/38),其中有7株(18.42%)同时耐3种药物。对阿米卡星、左氧氟沙星、四环素、呋喃妥因、甲氨苄啶/磺胺甲恶唑、庆大霉素耐药率为23.68%~44.74%,阿莫西林/克拉维酸、头孢唑啉、头孢他啶、头孢曲松、头孢吡肟、氨曲南、环丙沙星耐药率均大于52.63%,氨苄西林、哌拉西林耐药率100%。有11 株肺炎克雷伯菌碳青霉烯酶表型检测为阳性,占28.95%(11/38),其中5株携带bla_KPC基因,均为KPC-2型;4株携带bla_NDM基因,均为NDM-1型。9株携带bla_KPC或bla_NDM耐药基因的菌株同源性分析显示有8种不同PFGE带型,5种MLST 型别(ST型)。5种ST型为ST11、ST15、ST395、ST1031、ST1412,其中以ST11为主。结论 肺炎克雷伯菌耐药及多重耐药现象严重,肺炎克雷伯菌耐碳青霉烯类药物的主要的原因是携带KPC-2或者NDM-1基因,菌株基因型多态性大,提示病例之间不存在关联性。     

利用MLST技术对浙江省大肠杆菌O157的分子流行病学研究

目的对浙江省2005-2010年大肠杆菌O157分离株进行分子分型研究,了解菌株间的遗传进化关系,为浙江省大肠杆菌O157监测及爆发疫情的控制提供基础。方法选择Pasteur大肠杆菌多位点序列分型（Multilocus SequenceTyping,MLST）方案,对大肠杆菌O157分离株及882364菌株进行MLST分型,确定菌株序列型（Sequence type,ST）;采用DNAsp、eBURST、START2等软件进行分析。结果 30株菌中,27株O157∶H7菌株具备相同序列型（ST-284）,占90%（27/30）,其他3株菌序列型分别为ST-367、ST-125、ST-296。8个管家基因核苷酸多态性（Pi）范围为0.00119（polB）～0.00648（uidA）,putP基因多态性位点比例最高（4.6%）,trpA基因多态性位点比例最低（0.4%）。进化分析结果显示,这些序列型分别属于Group 1及Group 11群。结论浙江省动物源性大肠杆菌O157∶H7的主要序列型为ST-284,与江苏省病人株882364（ST-296）具有同一个进化祖先,提示应加强浙江省大肠杆菌O157病原学监测。     

河北省B群脑膜炎奈瑟菌药物敏感性及分子分型分析

目的了解河北省分离的9株B群脑膜炎奈瑟菌（Neisseriameningitides，Nm）的药物敏感性及分子流行病学特征。方法对分离菌株进行E—test法药物敏感性试验和多位点序列分型（MultilocusSequenceTyping，MLST）分析。结果药敏试验显示，9株B群Nm均对磺胺类抗生素耐药；6株分离自健康人的Nm中2株对喹诺酮类抗生素耐药；3株分离自流脑病人的Nm中的2株菌株对喹诺酮类抗生素耐药，且对青霉素类抗生素的敏感性降低。显示9株Nm菌株中的6株属于ST-4821克隆群，1株属于ST-175克隆群，2株无相应的克隆群。结论高致病性ST-4821已成为河北省B群Nrn的优势克隆群，提示应加强流脑的病原学监测。     

4株携带blaNDM基因的弗氏柠檬酸杆菌的分子特征研究

目的 研究携带blaNDM基因的耐碳青霉烯类弗氏柠檬酸杆菌的分子特征，为临床治疗提供参考。方法 收集宁夏某医院患者感染的耐碳青霉烯类弗氏柠檬酸杆菌并进行药敏试验，扩增菌株耐药基因，验证细菌表型，分析blaNDM基因水平转移情况，应用多位点序列分型进行同源性分析。结果 4株菌主要来自肝胆外科，对碳青霉烯类抗菌药物高度耐药，各菌株均检出blaNDM基因和其他不同耐药基因，碳青霉烯酶表型阳性，4株菌成功进行质粒接合试验，blaNDM基因均转移至大肠埃希菌J53AZ^R,MLST分型显示共4种ST型，分别为ST85、ST116、ST131和ST551。结论 检出4株携带blaNDM基因的耐碳青霉烯类弗氏柠檬酸杆菌，菌株对多种抗生素耐药，临床应加强抗生素使用管理，医院应重视耐药菌检测和监测工作的实施，防止耐药菌进一步流行造成更大危害。     

2014-2019年阜阳市食品和病人分离单增李斯特菌的分子特征分析

目的 比较分析阜阳市食品和病人分离单增李斯特菌的毒力基因、分子型别,建立阜阳市单增李斯特菌分子特征信息数据库,为防控单增李斯特菌病提供科学依据。方法 对阜阳市2014-2019年分离自食品和病人的55株单增李斯特菌,用聚合酶链反应(PCR)检测其毒力基因prfA、plcB、hly、actA、iap、inlA;用脉冲场凝胶电泳(PFGE)技术进行分子分型及同源性分析;用多位点序列分型(MLST)技术进行分子分型和聚类分析。结果 55株单增李斯特菌全部携带prfA、plcB、hly、actA、iap、inlA基因;PFGE将其分为21个带型,相似度60.3%~100%;MLST将其分为14个ST型,ST9型为优势型别;3株病人分离菌株的PFGE带型和ST型互不相同,其中2株病人来源菌株分别与食品分离株具有相同PFGE带型和ST型。结论 阜阳市食品和病人中分离的单增李斯特菌均携带6个毒力基因,食品分离菌株的分子型别呈现出多样性,其中存在同型别单增李斯特菌持续性污染现象,病人分离菌株具有与食品来源菌株相同的PFGE带型和ST型,提示食源性感染的风险较高。     

内蒙古布鲁氏菌临床分离株多位点序列分型研究

目的探讨临床分离布鲁氏菌的种群结构和遗传进化特征。方法布鲁氏菌标准参考菌株羊种16M,牛种544A和猪种1 330 S作为对照菌株,采用多位点序列分型(Multiple locus sequence typing,MLST)方法对内蒙古地区分离的116株羊种布鲁氏菌进行分析,确定待测菌株的序列型(STs),用软件BioNumerics(Version 5.0)构建菌株的最小生成树。结果116株羊种布鲁氏菌全部为ST8型,9个MLST位点的变异完全相同,分离株种群结构单一;羊种菌的生物型与ST型无明显关联,ST8型菌系该地区的主要流行菌种,并与内蒙古地区先前分离羊种布鲁氏菌进化程度相同,且有密切的亲缘关系。结论羊种布鲁氏菌的序列分型(ST)与常规生物分型存在差异,ST8型菌可能具有较强的环境适应性和致病性。MLST是一种较好的羊布鲁氏菌种群和进化关系研究方法,但更适合于具有较大时间跨度和地理分布菌株间的流行病学调查。     

首次自褐家鼠粪便标本中检出香港海鸥菌

目的了解褐家鼠粪便标本中是否携带香港海鸥菌,并分析其耐药特征及分子分型。方法2015年6月-2016年5月,利用笼捕法于广州市南方医院及其周边居民区捕获褐家鼠,其粪便样本经增菌后接种改良头孢哌酮MacConkey琼脂(CMA)平板培养分离可疑菌株,经生化试验及16SrRNA测序确认。采用K-B法进行药物敏感性测定,并运用多位点序列分型进行分子分型分析。结果共捕获褐家鼠191只,并在2份褐家鼠粪便标本中检出香港海鸥菌,检出率为1.05%。两株菌16SrRNA的测序结果与香港海鸥菌标准株HKU1的一致性达100%。药敏试验显示两株分离株均对头孢菌素类抗生素及利福平耐药。多位点序列分型结果显示两株菌分别为2个新的ST型:ST-163和ST-164。结论褐家鼠粪便中存在香港海鸥菌污染。褐家鼠与人类生活关系密切,可能为人类感染的的另一潜在来源。     

苏州市空肠弯曲菌耐药与分子特征分析

目的 了解苏州市空肠弯曲菌的耐药情况和分子特征。方法 对2018年-2022年苏州市分离的空肠弯曲菌进行全基因组测序分析了解毒力基因、耐药基因、多位点序列分型(Multi-locus sequence typing, MLST)等分子特征,通过琼脂稀释法了解其耐药表型。结果 61株空肠弯曲菌被分为42个ST型别,以ST464占比最高(9.8%),包含12个CC群,以CC21占比最高(19.7%)。61株菌共检出18种耐药基因,97种毒力基因。药敏试验耐药率排前3位的抗生素分别是萘啶酸,四环素和环丙沙星,耐药率分别为90.2%,88.5%和73.8%,对红霉素100.0%敏感,多重耐药率达到41.0%。结论 苏州市空肠弯曲菌多重耐药率高,携带耐药基因和毒力基因较多,克隆群以CC21为主,但ST型别较为分散,呈现较高的遗传多样性。     

From the Cover: INAUGURAL ARTICLE by a Recently Elected Academy Member:Is there tree senescence? The fecundity evidence

Despite its importance for forest regeneration, food webs, and human economies, changes in tree fecundity with tree size and age remain largely unknown. The allometric increase with tree diameter assumed in ecological models would substantially overestimate seed contributions from large trees if fecundity eventually declines with size. Current estimates are dominated by overrepresentation of small trees in regression models. We combined global fecundity data, including a substantial representation of large trees. We compared size–fecundity relationships against traditional allometric scaling with diameter and two models based on crown architecture. All allometric models fail to describe the declining rate of increase in fecundity with diameter found for 80% of 597 species in our analysis. The strong evidence of declining fecundity, beyond what can be explained by crown architectural change, is consistent with physiological decline. A downward revision of projected fecundity of large trees can improve the next generation of forest dynamic models.

“Belgium, Luxembourg, and The Netherlands are characterized by “young” apple orchards, where over 60% of the trees are under 10 y old. In comparison, Estonia and the Czech Republic have relatively “old” orchard[s] with almost 60% and 43% over 25 y old” (1).

“The useful lives for fruit and nut trees range from 16 years (peach trees) to 37 years (almond trees)…. The Depreciation Analysis Division believes that 61 years is the best estimate of the class life of fruit and nut trees based on the information available” (2).

When mandated by the 1986 Tax Reform Act to depreciate aging orchards, the Office of the US Treasury found so little information that they ultimately resorted to interviews with individual growers (2). One thing is clear from the age distributions of fruit and nut orchards throughout the world (1, 3, 4): Standard practice often replaces trees long before most ecologists would view them to be in physiological decline, despite the interruption of profits borne by growers as transplants establish and mature. Although seed establishment represents the dominant mode for forest regeneration globally, and the seeds, nuts, and fruits of woody plants make up to 3% of the human diet (5, 6), change in fecundity with tree size and age is still poorly understood. We examine here the relationship between tree fecundity and diameter, which is related to tree age in the sense that trees do not shrink in diameter (cambial layers typically add a new increment annually), but growth rates can range widely. Still, it is important not to ignore the evidence that declines with size may also be caused by aging. Although most analyses do not separate effects of size from age (because age is often unknown and confounded with size), both may contribute to size–fecundity relationships (7). Grafting experiments designed to isolate extrinsic influences (size and/or environment) from age-related gene expression suggest that size alone can sometimes explain declines in growth rate and physiological performance (8–10), consistent with pruning/coppicing practice to extend the reproductive life of commercial fruit trees. Hydraulic limitation can affect physiological function, including reduced photosynthetic gain that might contribute to loss of apical dominance, or “flattening” of the crown with increasing height (11–16). The slowing of height growth relative to diameter growth in large trees is observed in many species (12, 17). At least one study suggests that age by itself may not lead to decline in fecundity of open-grown, generally small-statured bristlecone pine (Pinus longaeva) (18). By contrast, some studies provide evidence of tree senescence, including age-related genetic changes in meristems of grafted scions that cause declines in physiological function (19–22). Koenig et al. (23) found that fecundity declined in the 5 y preceding death in eight Quercus species, although cause of death here, as in most cases, is hard to identify. Fielding (24) found that cone size of Pinus radiata declines with tree age and smaller cones produce fewer seeds (25). Some studies support age-related fecundity declines in herbaceous species (26–28). Thus, there is evidence to suggest the fecundity schedules might show declines with size, age, or both.The reproductive potential of trees as they grow and age is of special concern to ecologists because, despite being relatively rare, large trees can contribute disproportionately to forest biomass due to the allometric scaling that amplifies linear growth in diameter to a volume increase that is more closely related to biomass (29, 30). Understanding the role of large trees can also benefit management in recovering forests (31). If allometric scaling applies to fecundity, then these large individuals might determine the species and genetic composition of seeds that compete for dominance in future forests.Unfortunately, underrepresentation of big trees in forests frustrates efforts to infer how fecundity changes with size. Simple allometric relationships between seed production and tree diameter can offer useful predictions for the small- to intermediate-size trees that dominate observational data, so it is not surprising that modeling began with the assumption of allometric scaling (32–36). Extrapolation from these models would predict that seed production by the small trees from which most observations come may be overwhelmed by big trees. Despite the increase with tree size assumed by ecologists (37), evidence for declining reproduction in large trees has continued to accumulate from horticultural practice (3, 4, 38, 39) and at least some ecological (40–45) and forestry literature (46, 47). However, we are unaware of studies that evaluate changes in fecundity that include substantial numbers of large trees.Understanding the role of size and age is further complicated by the fact that tree fecundity ranges over orders of magnitude from tree to tree of the same species and within the same tree from year to year—a phenomenon known as “masting.” The variation in seed-production data requires large sample sizes not only to infer the effects of size, but also to account for local habitat and interannual climate variation. For example, a one-time destructive harvest to count seeds in felled trees (48, 49) misses the fact that the same trees would offer a different picture had they been harvested in a different year. An oak that produces 100 acorns this year may produce 10,000 next year. A pine that produces 500 cones this year can produce zero next year. Few datasets offer the sample sizes of trees and tree years needed to estimate effects of size and habitat conditions in the face of this high intertree and interyear variability (43).We begin this analysis by extending allometric scaling to better reflect the geometry of fecundity with tree size. We then reexamine the size–fecundity relationship using data from the Masting Inference and Forecasting (MASTIF) project (50), which includes substantial representation of large trees, and a modeling framework that allows for the possibility that fecundity plateaus or even declines in large trees. Unlike previous studies, we account for the nonallometric influences that come through competition and climate. We demonstrate that fecundity–diameter relationships depart substantially from allometric scaling in ways that are consistent with physiological senescence.Continuous increase with size has been assumed in most models of tree fecundity, supported in part by allometric regressions against diameter, typically of the formlogMf=β0+βD⁡log⁡D[1]for fecundity mass Mf=m×f (48, 51), where D is tree diameter, m is mass per seed, and fecundity f is seeds per tree per year. Of course, this model cannot be used to determine whether or how fecundity changes with tree diameter unless expanded to include additional quadratic or higher-order terms (52).The assumption of continual increase in fecundity was interpreted from early seed-trap studies, which initially assumed that βD=2, i.e., fecundity proportional to stem basal area (33−34, 51). Models subsequently became more flexible, first with βD values fitted, rather than fixed, yielding estimates in the range (0.3, 0.9) in one study (ref. 52, 18 species) and (0, 4.1) in another (ref. 56, 4 species). However, underrepresentation of large trees in typical datasets means that model fitting is dominated by the abundant small size classes.To understand why data and models could fail to accurately represent change in fecundity with size, consider that allometric scaling in Eq. 1 can be maintained dynamically only if change in both adheres to a strict proportionality1fdfdt∝1DdDdt[2](57). For allometric scaling, any variable that affects diameter growth has to simultaneously affect change in fecundity and in the same, proportionate way. In other words, allometric scaling cannot hold if there are selective forces on fecundity that do not operate through diameter growth and vice versa.On top of this awkward constraint that demands proportionate responses of growth and fecundity, consider further that standard arguments for allometric scaling are not directly relevant for tree fecundity. Allometry is invoked for traits that maintain relationships between body parts as an organism changes size (29). For example, a diameter increment translates to an increase in volume throughout the tree (58, 59). Because the cambial layer essentially blankets the tree, a volume increment cannot depart much from a simple allometric relationship with diameter. However, the same cannot be said for all plant parts, many of which clearly do not allometrically scale; for example, seed size does not scale with leaf size (60), presumably because structural constraints are not the dominant forces that relate them (61).To highlight why selective forces might not generate strict allometric scaling for reproduction, consider that a tree allocates a small fraction of potential buds to reproduction in a given year (62, 63). Still, if the number of buds on a tree bears some direct relationship to crown dimensions and, thus, diameter, there might be allometric scaling. However, the fraction of buds allocated to reproduction and their subsequent development to seed is affected by interannual weather and other selective forces (e.g., bud abortion, pollen limitation) in ways that diameter growth is not (64–66). In fact, weather might have opposing effects on growth and reproduction (67). Furthermore, resources can change the relationship between diameter and fecundity, including light levels (52, 68–70) and atmospheric CO2 (71).Some arguments based on carbon balance anticipate a decline in fecundity with tree size (72). Increased stomatal limitation (11) and reduced leaf turgor pressure (14, 73) from increasing hydraulic path length could reduce carbon gains in large trees. Assimilation rates on a leaf area basis can decline with tree size (74), while respiration rate per leaf area can increase [Sequoia sempervirens (75), Liquidambar styraciflua (76), and Pinus sylvestris (77)], consistent with the notion that whole-plant respiration rate may roughly scale with biomass (78). Maintenance respiration costs scale with diameter in some tropical species (79) but perhaps not in Pinus contorta and Picea engelmannii (80). Self-pruning of lower branches can reduce maintenance costs (81), but the ratio of carbon gain to respiration cost can still decline with size, especially where leaf area plateaus and per-area assimilation rates of leaves decline in large trees.The question of size–fecundity relationships is related indirectly to the large literature on interannual variation in growth–fecundity allocation (3, 4, 43, 67, 82–87). The frequency and timing of mast years and species differences in the volatility of seed production can be related to short-term changes in physiological state and pollen limitation that might not predict the long-term relationships between size and reproductive effort. The interannual covariance in diameter growth and reproductive effort can range from strong in some species to weak in others (70, 87, 88). Understanding the relationships between short-term allocation and size–fecundity differences will be an important focus of future research.Estimating effects of size on fecundity depends on the distribution of diameter data, [D], where the bracket notation indicates a distribution or density. For some early-successional species, the size distribution changes from dominance by small trees in young stands to absence of small trees in old stands. If our goal was to describe the population represented by a forest inventory plot, we would typically think about the joint distribution of fecundity and diameter values, [f,D]=[f|D][D], that is represented by the sample. The size–fecundity relationship estimated for a stand at different successional stages would diverge simply due to the distribution of diameters, i.e., differences in [D]. For example, application of Eq. 1 to harvested trees selected to balance size classes (uniform [D]) (48) overpredicts fecundity for large trees (49), but the relevance of such regressions for natural stands, where large trees are often rare, is unclear. Studies that expand Eq. 1 to allow for changing relationships with tree size now provide increasing evidence for a departure from allometric scaling in large trees (43, 70), despite dominance by small- to intermediate-size trees in these datasets. Here our goal is to understand the size–fecundity relationship [f|D] as an attribute of a species, i.e., not tied to a specific distribution of size classes observed in a particular stand.The well-known weak relationship between tree size and age that comes from variable growth histories makes it important to clarify the implications of any finding of fecundity that declines with tree size: Can it happen if there are not also fecundity declines with tree age? The only argument for continuing increase in fecundity with age in the face of observed decreases with size would have to assume that the biggest trees are also the youngest trees. Of course, a large individual can be younger than a small individual. However, at the species level, integrating over populations sampled widely, mean diameter increases with age; at the species level, declines with size also imply declines with age. Estimating accurate species-level size effects requires distributed data and large sample sizes. The analysis here fits species-level parameters, with 585,670 trees and 10,542,239 tree years across 597 species.Phylogenetic analysis might provide insight into the pervasiveness of fecundity declines with size. Inferring change in fecundity with size necessarily requires more information than is needed to fit a single slope parameter βD in the simple allometric model. The noisier the data, the more difficult it becomes to estimate the additional parameters that are needed to describe changes in the fecundity relationship with size. We thus expect that noise alone will preclude finding size-related change in some species, depending on sample size and non–size-related variation. If the vagaries of noisy data and the distribution of diameters preclude estimation of declines in some species, then we do not expect that phylogeny will explain which species do and do not show these declines. Rather than phylogeny, this explanation would instead be tied to sample size and the distribution of diameter data. Conversely, phylogenetic conservatism, i.e., a tendency for declines to be clustered in related species, could suggest that fecundity declines are real.To understand how seed production changes with tree size, our approach combines theory and data to evaluate allometric scaling and the alternative that fecundity may decline in large trees, consistent with physiological decline and senescence. We exploit two advances that are needed to determine how fecundity scales with tree size. First, datasets are needed with large trees, because studies in the literature often include few or none (85, 89, 90). Second, methods are introduced that are flexible to the possibility that fecundity continues to increase with size or not. We begin with a reformulation of allometric scaling, recognizing that change in fecundity could be regulated by size, without taking the form of Eq. 1 (Materials and Methods and SI Appendix, section S2). In other words, there could be allometric scaling with diameter, but it is not the relationship that has been used for structural quantities like biomass. We then analyze the relationships in data using a model that not only allows for potential changes in fecundity with size, but at the same time accounts for self-shading and shading by neighbors and for environmental variables that can affect fecundity and growth (Materials and Methods and SI Appendix, section S3). The fitted model is compared with our expanded allometric model to identify potential agreement. Finally, we examined phylogenetic trends in the species that do and do not show declines.     

Phylogenomics with paralogs

Phylogenomics heavily relies on well-curated sequence data sets that comprise, for each gene, exclusively 1:1 orthologos. Paralogs are treated as a dangerous nuisance that has to be detected and removed. We show here that this severe restriction of the data sets is not necessary. Building upon recent advances in mathematical phylogenetics, we demonstrate that gene duplications convey meaningful phylogenetic information and allow the inference of plausible phylogenetic trees, provided orthologs and paralogs can be distinguished with a degree of certainty. Starting from tree-free estimates of orthology, cograph editing can sufficiently reduce the noise to find correct event-annotated gene trees. The information of gene trees can then directly be translated into constraints on the species trees. Although the resolution is very poor for individual gene families, we show that genome-wide data sets are sufficient to generate fully resolved phylogenetic trees, even in the presence of horizontal gene transfer.Molecular phylogenetics is primarily concerned with the reconstruction of evolutionary relationships between species based on sequence information. To this end, alignments of protein or DNA sequences are used, whose evolutionary history is believed to be congruent to that of the respective species. This property can be ensured most easily in the absence of gene duplications and horizontal gene transfer (HGT). Phylogenetic studies judiciously select families of genes that rarely exhibit duplications (such as rRNAs, most ribosomal proteins, and many of the housekeeping enzymes). In phylogenomics, elaborate automatic pipelines such as HaMStR (1), are used to filter genome-wide data sets to at least deplete sequences with detectable paralogs (homologs in the same species).In the presence of gene duplications, however, it becomes necessary to distinguish between the evolutionary history of genes (gene trees) and the evolutionary history of the species (species trees) in which these genes reside. Leaves of a gene tree represent genes. Their inner nodes represent two kinds of evolutionary events, namely the duplication of genes within a genome—giving rise to paralogs—and speciations, in which the ancestral gene complement is transmitted to two daughter lineages. Two genes are (co)orthologous if their last common ancestor in the gene tree represents a speciation event, whereas they are paralogous if their last common ancestor is a duplication event; see refs. 2 and 3 for a more recent discussion on orthology and paralogy relationships. Speciation events, in turn, define the inner vertices of a species tree. However, they depend on both the gene and the species phylogeny, as well as the reconciliation between the two. The latter identifies speciation vertices in the gene tree with a particular speciation event in the species tree and places the gene duplication events on the edges of the species tree. Intriguingly, it is nevertheless possible in practice to distinguish orthologs with acceptable accuracy without constructing either gene or species trees (4). Many tools of this type have become available over the last decade; see refs. 5 and 6 for a recent review. The output of such methods is an estimate Θ of the true orthology relation Θ^?, which can be interpreted as a graph G_Θ whose vertices are genes and whose edges connect estimated (co)orthologs.Recent advances in mathematical phylogenetics suggest that the estimated orthology relation Θ contains information on the structure of the species tree. To make this connection, we combine here three abstract mathematical results that are made precise in Materials and Methods below.

• i)Building upon the theory of symbolic ultrametrics (7), we showed that in the absence of horizontal gene transfer, the orthology relation of each gene family is a cograph (8). Cographs can be generated from the single-vertex graph K₁ by complementation and disjoint union (9). This special structure of cographs imposes very strong constraints that can be used to reduce the noise and inaccuracies of empirical estimates of orthology from pairwise sequence comparison. To this end, the initial estimate of G_Θ is modified to the closest correct orthology relation G_Θ^? in such a way that a minimal number of edges (i.e., orthology assignments) are introduced or removed. This amounts to solving the cograph-editing problem (10, 11).
• ii)It is well known that each cograph is equivalently represented by its cotree (9). The cotree is easily computed for a given cograph. In our context, the cotree of G_Θ^? is an incompletely resolved event-labeled gene tree. That is, in addition to the tree topology, we know for each internal branch point whether it corresponds to a speciation or a duplication event. Even though adjacent speciations or adjacent duplications cannot be resolved, the tree faithfully encodes the relative order of any pair of duplication and speciation (8). In the presence of horizontal gene transfer, G_Θ may deviate from the structural requirements of a cograph. Still, the situation can be described in terms of edge-colored graphs whose subgraphs are cographs (7, 8), so that the cograph structure remains an acceptable approximation.
• iii)Every triple (rooted binary tree on three leaves) in the cotree that has leaves from three species and is rooted in a speciation event also appears in the underlying species tree (12). Thus, the estimated orthology relation, after editing to a cograph and conversion to the equivalent event-labeled gene tree, provides much information on the species tree. This result allows us to collect, from the cotrees for each gene family, partial information on the underlying species tree. Interestingly, only gene families that harbor duplications, and thus have a nontrivial cotree, are informative. If no paralogs exist, then the orthology relation G_Θ is a clique (i.e., every family member is orthologous to every other family member) and the corresponding cotree is completely unresolved, and hence contains no triple. On the other hand, full resolution of the species tree is guaranteed if at least one duplication event between any two adjacent speciations is observable. The achievable resolution therefore depends on the frequency of gene duplications and the number of gene families.

Despite the variance reduction due to cograph editing, noise in the data, as well as the occasional introduction of contradictory triples as a consequence of horizontal gene transfer, is unavoidable. The species triples collected from the individual gene families thus will not always be congruent. A conceptually elegant way to deal with such potentially conflicting information is provided by the theory of supertrees in the form of the largest set of consistent triples (13, 14). The data will not always contain a sufficient set of duplication events to achieve full resolution. To this end, we consider trees with the property that the contraction of any edge leads to the loss of an input triple. There may be exponentially many alternative trees of this type. They can be listed efficiently using Semple’s algorithms (15). To reduce the solution space further, we search for a least resolved tree in the sense of ref. 16, i.e., a tree that has the minimum number of inner vertices. It constitutes one of the best estimates of the phylogeny without pretending a higher resolution than actually supported by the data. In SI Appendix, we discuss alternative choices.The mathematical reasoning summarized above, outlined in Materials and Methods, and presented in full detail in SI Appendix, directly translates into a computational workflow, Fig. 1. It entails three NP-hard combinatorial optimization problems: cograph editing (11), maximal consistent triple set (17–19), and least resolved supertree (16). We show here that they are nevertheless tractable in practice by formulating them as Integer Linear Programs (ILP) that can be solved for both artificial benchmark data sets and real-life data sets, comprising genome-scale protein sets for dozens of species, even in the presence of horizontal gene transfer.Open in a separate windowFig. 1.Outline of the computational framework. Starting from an estimated orthology relation Θ, its graph representation G_Θ is edited to obtain the closest cograph G_Θ^*, which, in turn, is equivalent to a (not necessarily fully resolved) gene tree T and an event labeling t. From (T, t), we extract the set ?? of all relevant species triples. As the triple set ?? need not be consistent, we compute the maximal consistent subset ??^? of ??. Finally, we construct a least resolved species tree from ??^?.     

Solubilización in vitro del aceite de árbol de té y primeros resultados de su efecto antifúngico en onicomicosis

IntroductionOnychomycosis is the main cause of nail alteration. Hepatotoxicity, interference and low adherence to pharmacological treatment are associated. Therefore, our objective was to assess the in vitro effectiveness of tea tree essential oil (less harmful) against main causative agents of these infections.Material and methodsTrichophyton rubrum and Trichophyton mentagrophytes were isolated and inoculated at a concentration of 3x10⁵ CFU / mL in potato agar dextrose and tea tree essential oil at different concentrations to assess its effect by counting colony forming units and radial growth.ResultsTrichophyton rubrum growth inhibition was obtained at concentrations higher than 0.04% of the essential tea tree oil (p = 0.004). In the case of Trichophyton mentagrophytes, inhibition was obtained at 0.02% (p = 0.017), and even complete inhibition at a final concentration of the oil at 0.07%.ConclusionsTea tree essential oil inhibits the in vitro growth of the fungus and may be a less harmful alternative to the onychomycosis treatment.     

Pyrolysis of Pruning Residues from Various Types of Orchards and Pretreatment for Energetic Use of Biochar

The routine pruning and cutting of fruit trees provides a considerable amount of biowaste each year. This lignocellulosic biomass, mainly in the form of branches, trunks, rootstocks, and leaves, is a potential high-quality fuel, yet often is treated as waste. The results of a feasibility study on biochar production by pyrolysis of residues from orchard pruning were presented. Three types of biomass waste were selected as raw materials and were obtained from the most common fruit trees in Poland: apple (AP), pear (PR), and plum (PL) tree prunings. Two heating rates and three final pyrolysis temperatures were applied. For the slow (SP) and fast pyrolysis (FP) processes, the heating rates were 15 °C/min and 100 °C/min, respectively. The samples were heated from 25 °C up to 400, 500, and 600 °C. Chemical analyses of the raw materials were conducted, and the pyrolysis product yields were determined. A significant rise of higher heating value (HHV) was observed for the solid pyrolysis products, from approximately 23.45 MJ/kg for raw materials up to approximately 29.52 MJ/kg for pyrolysis products at 400 °C, and 30.53 MJ/kg for pyrolysis products at 600 °C. Higher carbon content was observed for materials obtained by fast pyrolysis conducted at higher temperatures.     

Half-century evidence from western Canada shows forest dynamics are primarily driven by competition followed by climate

Tree mortality, growth, and recruitment are essential components of forest dynamics and resiliency, for which there is great concern as climate change progresses at high latitudes. Tree mortality has been observed to increase over the past decades in many regions, but the causes of this increase are not well understood, and we know even less about long-term changes in growth and recruitment rates. Using a dataset of long-term (1958–2009) observations on 1,680 permanent sample plots from undisturbed natural forests in western Canada, we found that tree demographic rates have changed markedly over the last five decades. We observed a widespread, significant increase in tree mortality, a significant decrease in tree growth, and a similar but weaker trend of decreasing recruitment. However, these changes varied widely across tree size, forest age, ecozones, and species. We found that competition was the primary factor causing the long-term changes in tree mortality, growth, and recruitment. Regional climate had a weaker yet still significant effect on tree mortality, but little effect on tree growth and recruitment. This finding suggests that internal community-level processes—more so than external climatic factors—are driving forest dynamics.Forests provide fundamental ecosystem services for sustaining the global environment, such as storing carbon and maintaining biodiversity. These services, however, are at risk for decline as evidence has increasingly shown that forests in many parts of the world are undergoing rapid changes (1–4). Climate at the regional or global scale is often presumed to be responsible for these changes (5–14), with surprisingly little attention being paid to the possible effects of endogenous processes despite the fact that competition is often an important force driving stand dynamics and succession (15–18). How climate change and competition interplay to affect the long-term change of demographic rates and what are their relative contributions to the change are unanswered questions (19, 20).We addressed these questions by compiling data from 1,680 permanent sample plots (PSPs) that are located in undisturbed natural forests across western Canada (Fig. 1). The trees in these plots, which cover a wide geographic region spanning 32° of longitude and 10° of latitude primarily in the boreal zone, were censused over a period from 1958 to 2009 (Fig. 1). Within each plot, all standing trees with diameter at breast height (DBH) ≥ 9 cm were tagged, recorded, and remeasured at irregular time intervals (mean = 10 y) (SI Appendix, Fig. S1). Plot sizes ranged from 0.04 ha to 0.81 ha (mean = 0.14 ha). To reduce possible impact of plot sizes on our analyses, only the plots with at least 50 trees at their first census were selected. The plots have been censused three to eight times (mean of four times). In total, these plots contained 320,878 living trees over the study period (SI Appendix, Table S1).Open in a separate windowFig. 1.Locations of 1,680 permanent sample plots (PSPs) in western Canada. Each dot stands for one PSP. Four colors (dark blue, red, pink, and light blue) were used to show the distribution of PSPs in each of four provinces: British Columbia (777), Alberta (563), Saskatchewan (290), and Manitoba (50). The background colors represent Canada’s ecozones.We analyzed the changes of tree demographic rates (mortality, growth, and recruitment rates) over time at the species, stand, and regional levels and by stand age, tree size, and plot elevation (Methods). Two major possible drivers of the changes, competition and regional climate, were considered in our analyses. To test the effect of competition on the demographic rates, we used stand basal area (BA), basal area of larger trees (BAL), and stand density index (SDI), all commonly used in forestry (6, 12) as indexes of competition. To assess the effect of climate change, we selected mean warmest month temperature (MWMT), mean coldest month temperature (MCMT), and mean annual precipitation (MAP). We incorporated both competition and climatic variables simultaneously in the models, rather than separately as previous studies did (6, 11), and considered possible interactions between competition and climatic variables (21) in the models.     

Endoscopic ultrasound-guided transesophageal cholangiodrainage and consecutive endoscopic transhepatic Wallstent insertion into a jejunal stenosis

In cases where the papilla of Vater is unreachable because of pyloric/duodenal stenosis, or a catheter cannot be introduced into the papilla, or with recurrent tumor growth, or after previous gastrointestinal surgery, percutaneous transhepatic cholangiodrainage (PTCD) is considered to be the therapeutic alternative in cholestasis. The purpose of this report was to demonstrate that endoscopic ultrasound (EUS)-guided transesophageal cholangiodrainage is a feasible alternative in patients who decline to undergo PTCD. A 67-year-old female patient with recurrent tumor growth at the hepaticojejunostomy 17 months after a formerly resected cholangiocarcinoma (pT3, pN0 (0/2), M0, G2, R0; extended right hemihepatectomy), cholangitis, and peritoneal carcinomatosis underwent an EUS-guided transesophageal procedure to obtain cholangiodrainage by (i) puncture of a branch of the biliary tree at the left hepatic site, (ii) insertion of a guide wire into the bile duct and the anastomosed jejunum using the rendezvous technique with endoscopic retrograde cholangiopancreatography (ERCP)/conventional endoscopy, (iii) transesophagohepatic placement of an 8.5-Fr. double pigtail catheter, and (iv) transhepatic placement of a Wallstent through the jejunal stenosis, resulting in complete alleviation of the biliary and jejunal obstruction. There were no severe complications such as perforation or bleeding and no stent occlusion within the patient's lifetime of more than 3 months. Death was related to progressive tumor growth. EUS-guided transesophageal cholangiodrainage, here described in combination with Wallstent placement, is a reasonable, feasible, and encouraging treatment alternative in selected patients where conventional ERCP or PTCD is not an option.     

地理信息系统(GIS)与空间分析在大山区血吸虫病传播与控制研究中的应用

目的探索四川省血吸虫病流行区人群感染、环境污染和地理因素之间的关系,并确定感染的高度危险区域、疫区及其影响范围,进而确定地理因素、社会因素和生物因素对血吸虫病传播的影响。方法以四川省西昌市血吸虫病防治实验区收集的资料为基础采用特殊分析法,缓冲地带确定法,网络分析法,相关和分类回归树法（CART）进行分析。结果在血吸虫病防治规划中建立了GIS系统,并在血吸虫病流行区得到了应用。确定血吸虫感染的高度危险区、疫区以及影响血吸虫病传播的地理、社会和生物因素,可以为制订血吸虫病的调查和防治策略提供依据。     

Prediction,dynamics, and visualization of antigenic phenotypes of seasonal influenza viruses

Human seasonal influenza viruses evolve rapidly, enabling the virus population to evade immunity and reinfect previously infected individuals. Antigenic properties are largely determined by the surface glycoprotein hemagglutinin (HA), and amino acid substitutions at exposed epitope sites in HA mediate loss of recognition by antibodies. Here, we show that antigenic differences measured through serological assay data are well described by a sum of antigenic changes along the path connecting viruses in a phylogenetic tree. This mapping onto the tree allows prediction of antigenicity from HA sequence data alone. The mapping can further be used to make predictions about the makeup of the future A(H3N2) seasonal influenza virus population, and we compare predictions between models with serological and sequence data. To make timely model output readily available, we developed a web browser-based application that visualizes antigenic data on a continuously updated phylogeny.Seasonal influenza viruses evade immunity in the human population through frequent amino acid substitutions in their hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins (1). To maintain efficacy, vaccines against seasonal influenza viruses need to be updated frequently to match the antigenic properties of the circulating viruses. To facilitate informed vaccine strain selection, the genotypes and antigenic properties of circulating viruses are continuously monitored by the World Health Organization (WHO) Global Influenza Surveillance and Response System (GISRS), with a substantial portion of the virological characterizations being performed by the WHO influenza Collaborating Centers (WHO CCs) (2).Antigenic properties of influenza viruses are measured in hemagglutination inhibition (HI) assays (3) that record the minimal antiserum concentration (titer) necessary to prevent crosslinking of red blood cells by a standardized amount of virus based on hemagglutinating units. An antiserum is typically obtained from a single ferret infected with a particular reference virus. For a panel of test viruses, the HI titer is determined by a series of twofold dilutions of each antiserum. An antiserum is typically potent against the homologous virus (the reference virus used to produce the antiserum), but higher concentrations (and hence lower titers) are frequently required to prevent hemagglutination by other (heterologous) test viruses. HI titers typically decrease with increasing genetic distance between reference and test viruses (1).Given multiple antisera raised against different reference viruses and a panel of test viruses, WHO CCs routinely measure the HI titers T_aβ of all combinations of test viruses a and sera β, resulting in a matrix of HI titers (see Fig. 1A). The HI titer of a test virus a using antiserum β raised against the reference virus b is typically standardized as H_aβ = log₂(T_bβ) ? log₂(T_aβ), i.e., the difference in the number of twofold dilutions between homologous and heterologous titer. Standardized log₂ titers from many HI assays can be visualized in two dimensions via multidimensional scaling—an approach termed “antigenic cartography” (4). Although standard cartography does not use sequence information, sequences have been used as priors for positions in a Bayesian version of multidimensional scaling (5). To infer contributions of individual amino acid substitutions to antigenic evolution, Harvey et al. and Sun et al. (6, 7) have used models that predict HI titer differences by comparing sequences of reference and test viruses.Open in a separate windowFig. 1.Antigenic data and models for HI titers. (A) A typical table reporting HI titer data. Each number in the table is the maximum dilution at which the antiserum (column) inhibited hemagglutination of red blood cells by a virus (row). The red numbers on the diagonal indicate homologous titers. A typical HI assay consists of all reciprocal measurements of the available antisera and reference viruses, and a number of test viruses that are measured against all antisera, but for which no homologous antiserum exists. To make measurements using different antisera comparable, we define standardized log-transformed titers H_aβ relative to the homologous titer. (B) Each HI titer between antiserum α and virus b can be associated with a path on the tree connecting the reference and test viruses a and b, respectively, indicated as a thick line. The tree model seeks to explain the antigenic differences as additive contributions of branches. (C) In the substitution model, the sum over branches on the tree is replaced by a sum of contributions of amino acid substitutions.Here, we show that antigenic properties of seasonal influenza viruses are accurately described by a model based on the phylogenetic tree structure of their HA sequences. We use the model to show that HI titers have a largely symmetric and tree-like structure that can be used to define an antigenic distance between viruses. We show that large-effect substitutions account for about half of the total antigenic change and that the effect of specific substitutions is dependent on the genetic background in which they occur. We further investigate the ability of HI measurements to predict dominant clades in the next influenza season. To visualize antigenic properties on the phylogenetic tree, we have integrated the models of antigenic distances and the raw HI titer data into nextflu.org—an interactive real-time tracking tool for influenza virus evolution (8).This comprehensive summary of HA sequences from past and current influenza viruses linked to their antigenic properties has the potential to inform vaccine strain selections and facilitate efforts to predict successful influenza lineages (9–13).     

树鼩寄生虫研究进展

树鼩是最接近灵长类的小型哺乳动物,是模拟人类疾病的最好替代者,因此被广泛应用于各个研究领域。但是病原生物的感染是影响实验结果的重要因素。本文就树鼩所感染的寄生虫种类、感染率等情况作一综述。