Primary thrombophilia in Mexico. V. A comprehensive prospective study indicates that most cases are multifactorial

Over a 36-month period, 46 consecutive Mexican mestizos with a clinical marker associated with a primary hypercoagulable state were prospectively assessed by searching for the sticky platelet syndrome (SPS), the activated protein C resistance (aPCR) phenotype, coagulation protein C activity and antigen, coagulation protein S, antithrombin III, plasminogen, tissue-type plasminogen activator activity, plasminogen activator inhibitor activity, plasminogen activator inhibitor type 1, IgG and IgM isotypes of antiphospholipid antibodies, homocysteine levels, the factor V gene Leiden, Cambridge, Hong Kong, and Liverpool mutations, the 677 C-->T mutation in the 5,10-methylenetetrahydrofolatereductase (MTHFR), and the G20210A polymorphism in the 3'-untranslated region of the prothrombin gene. Of the 46 consecutive patients prospectively accrued in the study, only 12 (26%) were males, the median age being 38 years (range 10-63 years). In only four individuals (8%) could we not record any abnormality. In 5/42 patients with abnormal results (12%), a single abnormality was recorded, whereas in the remaining 37, two to five co-existing abnormalities were identified. We found 22 (48%) patients with the SPS, 11 (24%) with the aPCR phenotype, 5 (11%) with the factor V Leiden mutation, 7 (15%) with the prothrombin gene mutation, 29 (63%) with the MTHFR gene mutation, 11 (24%) with the factor V HR2 haplotype, 11 (24%) with antiphospholipid antibodies, 4 (9%) with PS deficiency, 6 (13%) with PC deficiency, one with the FV Hong Kong mutation, and one with AT-III deficiency. The results are consonant with the idea that most cases of thrombophilia in Mexico are multifactorial.     

Oto-palato-digital syndrome type II in two unrelated boys

We report on two boys with oto-palato-digital syndrome type II, characterized by growth retardation, bowed long bones, missing or hypoplastic fibulae, sclerosis of the skull base and wavy, irregular clavicles and ribs. The facial appearance is distinctive due to prominent forehead, widely spaced eyes, antimongologid slant of palpebral fissures, flattened nasal bridge and retrogenia. The mother of one patient showed a mild manifestation of oto-palato-digital syndrome type II. Only about 20 cases of this rare X-linked disorder have been reported so far. The similiarities and dissimilarities to oto-palato-digital syndrome type I are discussed.     

Apparently changing patterns of inheritance in Alport's hereditary nephritis: Genetic heterogeneity versus altered diagnostic criteria

With the use of more stringent diagnostic criteria, it has recently been shown that some large pedigrees of Alport's and non-Alport's hereditary nephritis fit sex-linked dominant inheritance (O'Neill et al. 1978). We have used similar diagnostic criteria and have studied a number of Michigan pedigrees in order to see if this hypothesis would be confirmed. We found one small pedigree which definitely shows male-to-male transmission, while one large pedigree is tentatively compatible with sex-linked dominant inheritance. Many of the other pedigrees suggested male-to-male transmission. This Michigan experience is compared to other published reports and found to be consistent, although a trend of fewer reports of male-to-male transmission is seen. We conclude that genetic heterogeneity of Alport's hereditary nephritis is likely.     

Clinical consequences of heterozygosity for autosomal-recessive diseases*,**

Heterozygotes of autosomal-recessive diseases can often be recognized by special heterozygote tests, since enzyme activities are normally reduced in comparison with the normal homozygote state. In Drosophila, the majority of recessive lethal mutations shows a reduction of fitness in heterozygotes, whereas in a strong minority fitness of heterozygotes is increased. This review will be devoted to a consideration of the extent to which heterozygotes for a wide variety of nominally recessive diseases are subject either to an increased liability for common diseases or slight shifts of behavioral characteristics. The available evidence has been collected and will be discussed in three steps: Most studies are available for phenylketonuria. For this group of diseases, a slight reduction of average--especially verbal--I.Q. in heterozygotes has been reported together with signs of a slightly increased cerebral irritability, a possible slight increase of risk for mental disease, and an increase of blood phenylalanine levels in stress situations. The PKU example is used to discuss methodological problems involved in such studies. Other conditions for which relevant deviations in heterozygotes are possible or even likely include among others lipid storage diseases, microcephaly, myoclonus epilepsy, Wilson's disease, galaktokinase deficiency, homocystinuria, recessive myotonia and ataxia- teleangiectasia (increased cancer risk). Since heterozygotes for autosomal recessive diseases are common, it is possible that an appreciable fraction of "multifactorial" genetic liabilities for common, "constitutional" or mental disease might simply be due to heterozygosity for genes whose homozygous affects are already well known. By the same token, much of the "normal" genetic variability influencing cognitive performance (I.Q.)--especially in the lower range--and personality characteristics could also be caused by recessive genes in the heterozygous state.     

Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM

The orchestrated action of genes controls complex biological phenotypes, yet the systematic discovery of gene and drug combinations that modulate these phenotypes in human cells is labor intensive and challenging to scale. Here, we created a platform for the massively parallel screening of barcoded combinatorial gene perturbations in human cells and translated these hits into effective drug combinations. This technology leverages the simplicity of the CRISPR-Cas9 system for multiplexed targeting of specific genomic loci and the versatility of combinatorial genetics en masse (CombiGEM) to rapidly assemble barcoded combinatorial genetic libraries that can be tracked with high-throughput sequencing. We applied CombiGEM-CRISPR to create a library of 23,409 barcoded dual guide-RNA (gRNA) combinations and then perform a high-throughput pooled screen to identify gene pairs that inhibited ovarian cancer cell growth when they were targeted. We validated the growth-inhibiting effects of specific gene sets, including epigenetic regulators KDM4C/BRD4 and KDM6B/BRD4, via individual assays with CRISPR-Cas–based knockouts and RNA-interference–based knockdowns. We also tested small-molecule drug pairs directed against our pairwise hits and showed that they exerted synergistic antiproliferative effects against ovarian cancer cells. We envision that the CombiGEM-CRISPR platform will be applicable to a broad range of biological settings and will accelerate the systematic identification of genetic combinations and their translation into novel drug combinations that modulate complex human disease phenotypes.New therapeutic strategies are needed to treat complex human diseases. Because disease phenotypes are often regulated by interwoven genetic networks, exploiting combination therapy to target multiple pathways, as opposed to only single ones, can enhance treatment efficacy (1). However, discovering effective combination therapies for human diseases is challenging with existing methods, due to the cost, effort, and labor required to construct and analyze each combination (2). For example, the National Cancer Institute tested ∼5,000 pairwise combinations of 100 cancer drugs against the NCI-60 panel in a study that took 2 y and cost about USD $4 million (3). Thus, there is a need for technological advances to accelerate the identification of effective combinatorial therapies. Here, we used our combinatorial genetics en masse (CombiGEM)-CRISPR platform to perform rapid pooled screening of pairwise genetic knockouts against genes coding for epigenetic regulators and then translated our screen hits into drug combinations against human ovarian cancer cells.CRISPR-Cas9 technology has been used for large-scale genetic perturbation screens with single-guide RNA (sgRNA) libraries for gene knockouts (4–7), repression, and activation (8, 9). Despite its simplicity for multiplexed genetic perturbations (10–12), new methods are needed to enable high-throughput CRISPR-Cas9–based screening with combinatorial sets of guide RNAs (gRNAs), which would be broadly useful for studying combinatorial gene functions in multigenic phenotypes and diseases. By using CombiGEM-based DNA assembly (13, 14), we developed a strategy for the simple and efficient assembly of barcoded combinatorial gRNA libraries. These libraries can be delivered into human cells by lentiviruses to create genetically ultradiverse cell populations harboring unique gRNA combinations that can be tracked via barcode sequencing in pooled assays. This strategy, termed CombiGEM-CRISPR, uses one-pot cloning steps to enable the assembly of combinatorial gRNA libraries, thus simplifying and accelerating the workflow toward systematic analysis of combinatorial gene functions.     

溃疡性结肠炎中药组方规律及整合药理学分子机制探索

目的通过中医传承辅助系统进行溃疡性结肠炎方剂经典名方和新方数据挖掘,运用整合药理学平台分析组方潜在作用分子靶点和信号通路。方法收集《中医方剂大辞典》治疗溃疡性结肠炎的组方,采用关联规则、复杂系统熵聚类等方法,确定各药物使用频次及关联规则,挖掘经典名方新方。应用整合平台靶标预测、网络构建等功能,获得经典名方和新方的成分-靶标网络、组方-溃疡性结肠炎相关靶标网络,通过功能分析和通路分析比较了经典名方与新方作用的溃疡性结肠炎相关靶标分子机制。结果对筛选出的257个处方进行分析,确定处方药物使用频次,药物之间关联规则,挖掘6个核心组合和3首新方,并进一步对比了经典名方和新方之间的可能分子机制差异。结论联用中医传承辅助系统以及整合药理学平台,可以从宏观和微观角度对溃疡性结肠炎的中药治疗挖掘经典名方和新方并研究潜在分子机制,为研究经典名方和衍生品提供了方向指导。     

朴炳奎解毒法治疗靶向所致皮疹的经验挖掘及应用研究

目的：通过数据挖掘的方法,探寻朴炳奎教授治疗肺癌靶向治疗患者及靶向治疗导致皮疹患者的用药规律,用客观数据总结其临床经验。方法：采用联机处理分析和复杂网络分析方法,对朴炳奎教授临床诊疗电子信息化模板的数据库中37例肺癌靶向治疗患者的病历资料进行回顾性分析(其中包含14例皮疹患者)。结果：通过数据挖掘,显示出朴师处方的核心思想以益气健脾为主,结合化痰祛湿、清热解毒为治则,体现出扶正与祛邪、辨病与辨证、局部与整体相结合的学术思想。结合专家访谈的形式,确立解毒消疹方的组成。结论：数据挖掘的结果与朴师治疗肺癌的扶正固本、祛邪解毒相结合的中西医结合学术思想得到了相互印证;确立了解毒消疹方的组成,即白芍、金银花、地肤子、蝉蜕。     

基于数据挖掘探讨全国名中医王行宽诊疗胸痹心痛遣方用药规律及学术经验

目的：运用中医传承计算平台V3.0软件,研究王行宽教授治疗胸痹心痛遣方用药规律,传承王行宽教授诊疗胸痹心痛的学术经验。方法：收集整理2017—2020年王行宽教授于湖南中医药大学门诊诊疗冠心病心绞痛患者的原始病历资料,录入中医传承计算平台V3.0,运用软件进行方药规律研究。结果：共收集王行宽教授治疗胸痹心痛处方1 044则,所用药物多为甘、苦药物,归经以肺经为主,其次为心、脾、肝、胃、肾经;所用方剂中使用最多的经方是生脉散,最高的经验方是心痛灵Ⅲ号方;高频数药物主要有麦冬、半夏、丹参、瓜蒌皮、黄连、五味子、柴胡等药物;药物的常用剂量多为3、5、10、15 g;组方规律分析得到常用药组合129个,置信度>0.99的组合有58个,并得到常见证型核心药物;药物聚类得到6个核心药物组合。结论：王行宽教授论治胸痹心痛以益气养营、豁痰化瘀、疏肝利胆为治疗思路,并根据胸痹心痛不同证型予以辨证施治,体现其“多脏调燮、综合治理”的学术思想,其核心处方可供临床从业者参考,但仍需要进一步的临床及实验研究验证其疗效。     

中药炮制学科和饮片产业传承与创新过程中几个值得探讨的问题

本文在近年来炮制学科和饮片产业的传承、创新、发展的基本状况的调研的基础上,通过对中药炮制学科和饮片产业传承与创新过程中几个值得探讨的问题的分析,从中药饮片的定义以及其传承、创新的内容、方式方面提出了几点见解。旨在抛砖引玉,求得共识,更进一步促进学科和行业的发展。