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1.
Klinfelter syndrome was first described in adult males with gynecomastia, azoospermia and hypergonadotropic hypogonadism. Children with the 47, XXY karyotype demonstrate few clinical findings so Klinefelter syndrome is rarely diagnosed until adult life. Besides children who have been diagnosed during prenatal genetic testing, in infancy a male with 47, XXY (or variants: 46, XY-47, XXY; 48, XXXY; 48, XXYY, 49, XXXXY) may be found while undergoing evaluation of micropenis, hypospadias, cryptorchidism or facial anomalies. The older child may present with learning disabilities, behavior disorders or tall stature. At the time of puberty, the clinical picture includes small testes, gynecomastia and an eunuchoid habitus. Early diagnosis of Klinefelter syndrome must be performed since it has been demonstrated that early treatment with androgens may ameliorate many aspects of the clinical symptoms and attenuate or prevent behavioral and psychiatric disorders associated with 47, XXY males.  相似文献   

2.
Our previous work using a melanoma progression model composed of melanocytic cells (melanocytes, primary and metastatic melanoma samples) demonstrated various deregulated genes, including a few known lncRNAs. Further analysis was conducted to discover novel lncRNAs associated with melanoma, and candidates were prioritized for their potential association with invasiveness or other metastasis‐related processes. In this sense, we found the intergenic lncRNA U73166 (ENSG00000230454) and decided to explore its effects in melanoma. For that, we silenced the lncRNA U73166 expression using shRNAs in a melanoma cell line. Next, we experimentally investigated its functions and found that migration and invasion had significantly decreased in knockdown cells, indicating an essential association of lncRNA U73166 for cancer processes. Additionally, using naïve and vemurafenib‐resistant cell lines and data from a patient before and after resistance, we found that vemurafenib‐resistant samples had a higher expression of lncRNA U73166. Also, we retrieved data from the literature that indicates lncRNA U73166 may act as a mediator of RNA processing and cell invasion, probably inducing a more aggressive phenotype. Therefore, our results suggest a relevant role of lncRNA U73166 in metastasis development. We also pointed herein the lncRNA U73166 as a new possible biomarker or target to help overcome clinical vemurafenib resistance.  相似文献   

3.
The cannabinoid 1 (CB1) allosteric modulator, 5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)-ethyl]-amide) (ORG27569), has the paradoxical effect of increasing the equilibrium binding of [3H](−)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxylpropyl]cyclohexan-1-ol (CP55,940, an orthosteric agonist) while at the same time decreasing its efficacy (in G protein-mediated signaling). ORG27569 also decreases basal signaling, acting as an inverse agonist for the G protein-mediated signaling pathway. In ligand displacement assays, ORG27569 can displace the CB1 antagonist/inverse agonist, N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide(SR141716A). The goal of this work was to identify the binding site of ORG27569 at CB1. To this end, we used computation, synthesis, mutation, and functional studies to identify the ORG27569-binding site in the CB1 TMH3-6-7 region. This site is consistent with the results of K3.28192A, F3.36200A, W5.43279A, W6.48356A, and F3.25189A mutation studies, which revealed the ORG27569-binding site overlaps with our previously determined binding site of SR141716A but extends extracellularly. Additionally, we identified a key electrostatic interaction between the ORG27569 piperidine ring nitrogen and K3.28192 that is important for ORG27569 to act as an inverse agonist. At this allosteric site, ORG27569 promotes an intermediate conformation of the CB1 receptor, explaining ORG27569''s ability to increase equilibrium binding of CP55,940. This site also explains ORG27569''s ability to antagonize the efficacy of CP55,940 in three complementary ways. 1) ORG27569 sterically blocks movements of the second extracellular loop that have been linked to receptor activation. 2) ORG27569 sterically blocks a key electrostatic interaction between the third extracellular loop residue Lys-373 and D2.63176. 3) ORG27569 packs against TMH6, sterically hindering movements of this helix that have been shown to be important for receptor activation.  相似文献   

4.
In this short report, the genome-wide homologous recombination events were re-evaluated for classical swine fever virus (CSFV) strain AF407339. We challenged a previous study which suggested only one recombination event in AF407339 based on 25 CSFV genomes. Through our re-analysis on the 25 genomes in the previous study and the 41 genomes used in the present study, we argued that there should be possibly at least two clear recombination events happening in AF407339 through genome-wide scanning. The reasons for identifying only one recombination event in the previous study might be due to the limited number of available CSFV genome sequences at that time and the limited usage of detection methods. In contrast, as identified by most detection methods using all available CSFV genome sequences, two major recombination events were found at the starting and ending zones of the genome AF407339, respectively. The first one has two parents AF333000 (minor) and AY554397 (major) with beginning and ending breakpoints located at 19 and 607 nt of the genome respectively. The second one has two parents AF531433 (minor) and GQ902941 (major) with beginning and ending breakpoints at 8397 and 11,078 nt of the genome respectively. Phylogenetic incongruence analysis using neighbor-joining algorithm with 1000 bootstrapping replicates further supported the existence of these two recombination events. In addition, we also identified additional 18 recombination events on the available CSFV strains. Some of them may be trivial and can be ignored. In conclusion, CSFV might have relatively high frequency of homologous recombination events. Genome-wide scanning of identifying recombination events should utilize multiple detection methods so as to reduce the risk of misidentification.  相似文献   

5.
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6.
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7.
We analyzed the temporal and spatial diversity of the microbiota in a low-usage and a high-usage hospital tap. We identified a tap-specific colonization pattern, with potential human pathogens being overrepresented in the low-usage tap. We propose that founder effects and local adaptation caused the tap-specific colonization patterns. Our conclusion is that tap-specific colonization represents a potential challenge for water safety.Humans are exposed to and consume large amounts of tap water in their everyday life, with the tap water microbiota representing a potent reservoir for pathogens (8). Despite the potential impact, our knowledge about the ecological diversification processes of the tap water microbiota is limited (4, 11).The aim of the present work was to determine the temporal and spatial distribution patterns of the planktonic tap water microbiota. We compared the summer and winter microbiota from two hospital taps supplied from the same water source. We analyzed 16S rRNA gene clone libraries by using a novel alignment-independent approach for operational taxonomic unit (OTU) designation (6), while established OTU diversity and richness estimators were used for the ecological interpretations.Tap water samples (1 liter) from a high-usage kitchen and a low-usage toilet cold-water tap in Akershus University Hospital, Lørenskog, Norway, were collected in January and July 2006. The total DNA was isolated and the 16S rRNA gene PCR amplified and sequenced. Based on the sequences, we estimated the species richness and diversity, we calculated the distances between the communities, and trees were constructed to reflect the relatedness of the microbiota in the samples analyzed. Details about these analytical approaches are given in the materials and methods section in the supplemental material.Our initial analysis of species composition was done using the RDPII hierarchical classifier. We found that the majority of pathogen-related bacteria in our data set belonged to the class Gammaproteobacteria. The genera encompassed Legionella, Pseudomonas, and Vibrio (Table ​(Table1).1). We found a significant overrepresentation of pathogen-related bacteria in the toilet tap (P = 0.04), while there were no significant differences between summer and winter samples. Legionella showed the highest relative abundance for the pathogen-related bacteria. With respect to the total diversity, we found that Proteobacteria dominated the tap water microbiota (representing 86% of the taxa) (see Table S1 in the supplemental material). There was, however, a large portion (56%) of the taxa that could not be assigned to the genus level using this classifier.

TABLE 1.

Cloned sequences related to human pathogensa
Sampling placeSampling timePathogenNCBI accession no.Identity (%)
ToiletSummerEscherichia coliEF41861499
ToiletSummerEscherichia sp.EF07430799
ToiletSummerLegionella sp.AY92415595
ToiletSummerLegionella sp.AY92415395
ToiletSummerLegionella sp.AY92415396
ToiletWinterLegionella sp.AY92406196
ToiletWinterLegionella sp.AY92415897
ToiletWinterLegionella sp.AY92415897
KitchenWinterLegionella sp.AY92399697
ToiletSummerPseudomonas fluorescensEF41307398
ToiletSummerPseudomonas fluorescensEF41307398
KitchenSummerPseudomonas fluorescensDQ20773199
ToiletWinterVibrio sp.DQ40838898
ToiletWinterVibrio sp.AB27476098
KitchenWinterVibrio sp.DQ40838898
KitchenWinterVibrio lentusAY29293699
KitchenWinterVibrio sp.AM18376597
ToiletWinterStenotrophomonas maltophiliaAY83773099
KitchenWinterStenotrophomonas maltophiliaDQ42487098
ToiletWinterStreptococcus suisAF28457898
ToiletWinterStreptococcus suisAF28457898
Open in a separate windowaThe relatedness between the cloned sequences and potential pathogens was determined by BLAST searches of the NCBI database, carried out using default settings.To obtain a better resolution of the uncharacterized microbiota, we analyzed the data using a clustering approach that is not dependent on a predefined bacterial group (see the materials and methods section in the supplemental material for details). These analyses showed that there were three relatively tightly clustered groups in our data set (Fig. ​(Fig.1A).1A). The largest group (n = 590) was only distantly related to characterized betaproteobacteria within the order Rhodocyclales. We also identified another large betaproteocaterial group (n = 320) related to Polynucleobacter. Finally, a tight group (n = 145) related to the alphaproteobacterium Sphingomonas was identified.Open in a separate windowFIG. 1.Tap water microbiota diversity, determined by use of a principal component analysis coordinate system. (A) Each bacterium is classified by coordinates, with the following color code: brown squares, kitchen summer; red diamonds, toilet summer; green triangles, kitchen winter; and green circles, toilet winter. (B and C) Each square represents a 1 × 1 (B) or 5 × 5 (C) OTU. PC1, first principal component; PC2, second principal component.The tap-specific distributions of the bacterial groups were investigated using density distribution analyses. A dominant population related to Polynucleobacter was identified for the toilet summer samples, while for the winter samples there was a dominance of the Rhodocyclales-related bacteria. The kitchen summer samples revealed a dominance of Sphingomonas. The corresponding winter samples did not reveal distinct high-density bacterial populations (see Table S2 in the supplemental material).Hierarchical clustering for the 1 × 1 OTU density distribution confirmed the relatively low overlap for the microbiota in the samples analyzed (Fig. ​(Fig.2).2). We found that the microbiota clustered according to tap and not season.Open in a separate windowFIG. 2.Hierarchical clustering for the density distribution of the tap water microbiota. The density of 1 × 1 OTUs was used as a pseudospecies for hierarchical clustering. The tree for the Cord distance matrix is presented, while the distances calculated using the three distance matrices Cord, Brad Curtis, and Sneath Sokal, respectively, are shown for each branch.We have described the species diversity and richness of the microbiota in Table S3 in the supplemental material. For the low taxonomic level, these analyses showed that the diversity and species richness were greater for the winter samples than for the summer samples. Comparing the two taps, the diversity and richness were greater in the kitchen tap than in the toilet tap. In particular, the winter sample from the kitchen showed great richness and diversity. The high taxonomic level, however, did not reveal the same clear differences as did the low level, and the distributions were more even. Rarefaction analyses for the low taxonomic level confirmed the richness and diversity estimates (see Fig. S1 in the supplemental material).Our final analyses sought to fit the species rank distributions to common rank abundance curves. Generally, the rank abundance curves were best fitted to log series or truncated log normal distributions (see Table S4 in the supplemental material). The log series distribution could be fit to all of the samples except the kitchen summer samples at the low taxonomic level, while the truncated log normal distribution could not be fit to the kitchen samples at the high taxonomic level. Interestingly, however, the kitchen winter sample was best fit to a geometric curve at both the high and the low taxonomic level.Diversifying, adaptive biofilm barriers have been documented for tap water bacteria (7), and it is known that planktonic bacteria can interact with biofilms in an adaptive manner (3). On the other hand, tap usage leads to water flowthrough and replacement of the global with the local water population by stochastic founder effects (1).Therefore, we propose that parts of the local diversity observed can be explained by local adaptation (10) and parts by founder effects (9).Most prokaryote diversity measures assume log normal or log series OTU dominance density distributions (5). The kitchen winter sample, however, showed deviations from these patterns by being correlated to geometric distributions (in addition to the log series and truncated log normal distributions for the high taxonomic level). This sample also showed a much greater species richness than the other samples. A possible explanation is that the species richness of the tap water microbiota can be linked to usage and that the kitchen tap is driven toward a founder microbiota by high usage.Since our work indicates an overrepresentation of Legionella in the low-usage tap, it would be of high interest to determine whether the processes for local Legionella colonization can be related to tap usage. Understanding the ecological forces affecting Legionella and other pathogens are of great importance for human health. At the Akerhus University Hospital, this was exemplified by a Pseudomonas aeruginosa outbreak in an intensive care unit, where the outbreak could be traced back to a single tap (2).  相似文献   

8.
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9.
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10.
Containment strategies for outbreaks of invasive Neisseria meningitidis disease are informed by serogroup assays that characterize the polysaccharide capsule. We sought to uncover the genomic basis of conflicting serogroup assay results for an isolate (M16917) from a patient with acute meningococcal disease. To this end, we characterized the complete genome sequence of the M16917 isolate and performed a variety of comparative sequence analyses against N. meningitidis reference genome sequences of known serogroups. Multilocus sequence typing and whole-genome sequence comparison revealed that M16917 is a member of the ST-11 sequence group, which is most often associated with serogroup C. However, sequence similarity comparisons and phylogenetic analysis showed that the serogroup diagnostic capsule polymerase gene (synD) of M16917 belongs to serogroup B. These results suggest that a capsule-switching event occurred based on homologous recombination at or around the capsule locus of M16917. Detailed analysis of this locus uncovered the locations of recombination breakpoints in the M16917 genome sequence, which led to the introduction of an ∼2-kb serogroup B sequence cassette into the serogroup C genomic background. Since there is no currently available vaccine for serogroup B strains of N. meningitidis, this kind capsule-switching event could have public health relevance as a vaccine escape mutant.  相似文献   

11.
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12.
We have previously shown that Fhit tumor suppressor protein interacts with Hsp60 chaperone machinery and ferredoxin reductase (Fdxr) protein. Fhit-effector interactions are associated with a Fhit-dependent increase in Fdxr stability, followed by generation of reactive oxygen species and apoptosis induction under conditions of oxidative stress. To define Fhit structural features that affect interactions, downstream signaling, and biological outcomes, we used cancer cells expressing Fhit mutants with amino acid substitutions that alter enzymatic activity, enzyme substrate binding, or phosphorylation at tyrosine 114. Gastric cancer cell clones stably expressing mutants that do not bind substrate or cannot be phosphorylated showed decreased binding to Hsp60 and Fdxr and reduced mitochondrial localization. Expression of Fhit or mutants that bind interactor proteins results in oxidative damage and accumulation of cells in G2/M or sub-G1 fractions after peroxide treatment; noninteracting mutants are defective in these biological effects. Gastric cancer clones expressing noncomplexing Fhit mutants show reduction of Fhit tumor suppressor activity, confirming that substrate binding, interaction with heat shock proteins, mitochondrial localization, and interaction with Fdxr are important for Fhit tumor suppressor function.Fhit protein is a powerful tumor suppressor that is frequently lost or reduced in cancer cells because of rearrangement of the exquisitely DNA damage-sensitive fragile FHIT gene. Restoration of Fhit expression suppresses tumorigenicity of cancer cells of various types, and the ability to induce apoptosis in cancer cells in vitro is reduced by specific Fhit mutations (1, 2).Through studies of signal pathways affected by Fhit expression, by searches for Fhit protein effectors, and by in vitro analyses of Fhit activity, we and others have defined Fhit enzymatic activity in vitro (3), apoptotic activity in cells and tumors (4–6), and most recently identification of a Fhit protein complex that affects Fhit stability, mitochondrial localization, and interaction with ferredoxin reductase (Fdxr)5 (7). The complex includes Hsp60 and Hsp10 that mediate Fhit stability and may affect import into mitochondria, where Fhit interacts with Fdxr, which is responsible for transferring electrons from NADPH to cytochrome P450 via ferredoxin. Virally mediated Fhit restoration in Fhit-deficient cancer cells increases production of intracellular reactive oxygen species (ROS), followed by increased apoptosis of cancer cells under oxidative stress conditions; conversely, Fhit-negative cells escape apoptosis, likely carrying oxidative DNA damage that contributes to accumulation of mutations.The Fhit protein sequence, showing high homology to the histidine triad (HIT) family of proteins, suggested that the protein product would hydrolyze diadenosine tetraphosphate or diadenosine triphosphate (Ap3A) (8), and in vitro studies showed that Ap3A was cleaved into ADP and AMP by Fhit. The catalytic histidine triad within Fhit was essential for catalytic activity (3), and a Fhit mutant that substituted Asn for His at the central histidine (H96N mutant) was catalytically inactive, although it bound substrate well (3). Early tumor suppression studies showed that cancer cells stably transfected with wild type (WT) or H96N mutant Fhit were suppressed for tumor growth in nude mice. This suggested the hypothesis that the Fhit-substrate complex sends the tumor suppression signal (9, 10). To test this hypothesis, a series of FHIT alleles was designed to reduce substrate-binding and/or hydrolytic rates and was characterized by quantitative cell-death assays on cancer cells virally infected with each allele. The allele series covered defects as great as 100,000-fold in kcat and increases as large as 30-fold in Km. Mutants with 2–7-fold increases in Km had significantly reduced apoptotic indices and the mutant with a 30-fold increase in Km retained little apoptotic function. Thus, the proapoptotic function of Fhit, which is likely associated with tumor suppressor function, is limited by substrate binding and is unrelated to substrate hydrolysis (11).Fhit, a homodimeric protein of 147 amino acids, is a target of tyrosine phosphorylation by the Src family protein kinases, which can phosphorylate Tyr-114 of Fhit in vitro and in vivo (12). After co-expression of Fhit with the Elk tyrosine kinase in Escherichia coli to generate phosphorylated forms of Fhit, unphosphorylated, mono-, and diphosphorylated Fhit were purified, and enzyme kinetics studies showed that monophosphorylated Fhit exhibited monophasic kinetics with Km and kcat values ∼2- and ∼7-fold lower, respectively, than for unphosphorylated Fhit. Diphosphorylated Fhit exhibited biphasic kinetics; one site had Km and kcat values ∼2- and ∼140-fold lower, respectively, than for unphosphorylated Fhit; the second site had a Km ∼60-fold higher and a kcat ∼6-fold lower than for unphosphorylated Fhit (13). Thus, it was possible that the alterations in Km and kcat values for phosphorylated forms of Fhit might favor formation and lifetime of the Fhit-Ap3A complex and enhance tumor suppressor activity (see
Fhit forms
Kinetic parameters
% Sub-G1
Direct binding
Subcellular location
Co-IP in vivo
8-OHdG
Apoptosis
Tumor suppressor
Km (mm)kcat (s–1)A549MKN74Hsp60FdxrHsp60Fdxr
Fhit WT 1.6 +/– 0.19 2.7 +/– 0.95 43 24 Yes Yes Cyt & mito Yes Yes Yes Yes Yes
Catalyt mutants
   H96D Up 2-fold Down >2 × 104 29 NT NT NT Cyt & mito Yes Yes NT Yes NT
   H96N Up 2-fold Down >5 × 105 31 14.4 NT NT Cyt & mito Yes Yes Yes Yes Yes
Loop mutants
   Y114A Up 23-fold Down 2-fold 3.7 NT NT NT Cyt +/– +/– +/– No No
   Y114D NT NT 2.9 6 NT NT Cyt +/– +/– – No –/+
   Y114E NT NT NT NT NT NT Cyt & mito –/+ –/+ – No NT
   Y114F Up 5-fold Up 1.1-fold 11.5 3 NT NT Cyt & mito –/+ –/+ – No No
   Y114W Up 5-fold Up 1.4-fold NT NT NT NT Cyt & mito –/+ – – NT NT
   del113–117 Up 10-fold Down 38-fold 5 NT NT NT NT NT NT – No NT
Other mutants
   L25W Up 7-fold Down 4-fold 15 NT NT NT Cyt – – – NT –/+
   I10W,L25W Up 32-fold Down 6-fold 11 NT NT NT NT NT NT NT NT NT
   F5W Up 3.3 fold NT NT 5 NT NT NT NT NT +/– No NT
Purified pFhit
   pFhit Down 0.4-fold Down 7-fold NA NA –/+ Yes NA NA NA NA NA NA
   ppFhit Down 0.4-fold Down > 100-fold NA NA –/+ Yes NA NA NA NA NA NA
Up 60-fold Down 6-fold
Open in a separate windowTo explore the in vivo importance of the Tyr-114 phosphorylation site and define Fhit-mediated signaling events, Semba et al. (14) compared the differential biological effects of Ad-FHIT WT and Ad-FHIT Tyr-114 mutant expression in human lung cancer cells. Caspase-dependent apoptosis was effectively induced only by WT Fhit protein. However, the biological significance of phosphorylation at Tyr-114 has been difficult to study because the endogenous phosphorylated forms have very short half-lives; activation of epidermal growth facto receptor family members induces Fhit phosphorylation by Src and proteasome degradation of phosphorylated Fhit (15).Although there are possible connections among the various pathways known to be altered in Fhit-deficient cells, apoptosis, DNA damage-response checkpoint activation, ROS production, and related biological effects of Fhit loss or overexpression, details of the pathway(s) leading from Fhit overexpression to cell death and tumor suppression have not been delineated. Now that a Fhit signaling complex has been identified, we set out to examine which structural features of Fhit protein might participate in individual steps of the pathway leading from Fhit overexpression through complex formation, subcellular localization, interaction with mitochondrial Fdxr, DNA damage induction, cell cycle changes, apoptosis, and ultimately tumor suppression. The underlying hypotheses were as follows: substrate-binding mutants would behave similarly to WT; nonsubstrate-binding mutants would be defective in some step of the pathway, perhaps complexing with heat shock proteins or Fdxr or perhaps induction of DNA damage; and Tyr-114 mutants, which also affect formation or stability of the enzyme-substrate complex, would also be defective in executing some step of the Fhit overexpression pathway to cell death. One goal was to identify specific mutants that exhibited deficiency in specific steps of the pathway, so that such mutants could be used to dissect each step in more detail. Using in vitro Fhit and Fhit-effector protein interactions, we aimed to determine the following: 1) which proteins of the complex interact directly with Fhit, and 2) the biological role of these interactions in vivo. Using cancer cells expressing exogenous WT and mutant Fhit proteins, we were able to examine the structural features of Fhit that affect the direct interaction with its effectors, participate in ROS production, and are necessary for tumor suppression activity.  相似文献   

13.
关于护士职业价值观文献回顾研究     
王玲  钱红花 《西北植物学报》2015,(6):70-72
通过对近些年关于护理人员职业价值观研究的回顾与总结,对关键词“价值观”“工作价值观”“职业价值观”“护士职业价值观”“护士工作价值观”“护生价值观”“护生职业价值观”“护生工作价值观”“医学生职业价值观”“组织价值观”“道德价值观”进行网络检索。针对护理人员职业价值观内涵的总结与界定,形成构成护理人员职业价值观因素分析,提出研究护理人员职业价值观时代新意。  相似文献   

14.
Huntington's disease: Dancing in a dish     
Kejing Zhang  Fei Yi  Guang-Hui Liu  Juan Carlos Izpisua Belmonte 《Cell research》2012,22(12):1627-1630
In a recent landmark paper, the Huntington''s disease (HD) iPSC Consortium reports on the establishment and characterization of a panel of iPSC lines from HD patients, and more importantly, the successful modeling of HD in vitro. In the same issue of Cell Stem Cell, An et al. reports on the successful targeted gene correction of HD in human iPSCs. Both advances are exciting, provide new resources for current and future HD research, and uncover new challenges to better understand and, most importantly, treat this devastating disease in the near future.Modeling human diseases using induced pluripotent stem cells (iPSCs) has created novel opportunities for both mechanistic studies as well as for the discovery of new disease therapies. Combined with advanced gene correction technology, human iPSCs hold great promise to provide patient-specific and mutation-free cells for potential cell replacement therapy. Huntington''s disease (HD) is an autosomal dominant neurodegenerative disorder, which causes motor dysfunction, psychiatric disturbances and cognitive impairment1. HD is caused by an expanded cystosine adenine guanine (CAG) tri-nucleotide repeat encoding polyglutamine in the first exon of the Huntingtin (HTT) gene. To date, there is no effective therapy for preventing the onset or slowdown of this disorder. Preliminary clinical trials using fetal neural grafts had shown long-lasting functional benefits in patients2. Though only effective in limited cases, these results suggest that cell-based therapy could be a potential treatment if a reliable and consistent cell source is available. For this purpose, an alternative cell source to overcome the logistical and biological hurdles of this disease had been actively explored in the past decade. With recent advancement in human iPSCs technology, HD patient-specific iPSCs coupled with an efficient directed cell differentiation protocol offers hope for an unlimited supply of autologous cells. Since HD is a monogenic disease, with a very well-established correlation between the number of CAG repeats and the age of disease onset, it provides an ideal target for iPSC-based gene correction that will allow for the production of disease-free cells for potential autologous cell therapy, and at the same time provide a much needed, valuable platform to further study the pathogenesis of the disease3,4.This is in fact what has been recently accomplished in two reports published in Cell Stem Cell5,6. The HD iPSC Consortium reports on the generation of HD patient-specific iPSC lines that showed CAG-repeat-expansion-associated phenotypes5. The study from An et al.6 reports on the successful targeted correction of expanded CAG repeat in HD patient iPSCs and the reversion of disease phenotypes.In the study reported from the HD iPSC Consortium, the authors generated 14 iPSC lines from HD patients and controls (listed in Open in a separate window
CodeNumber of iPSC lineCAG repeatsHD typeAge of sample procuredReprogramming strategyPhenotype detected cell typeGene correction line availablePhenotypeReferences
HD 43139/43Adult onset HD44 yearsOSKM (lentivirus)iPSCsnoIncreased Iysosomal activity7
HD 44442/44Adult onset HD59 years2 lines:OSKM (lentivirus) 2 lines: OSK (lentivirus)iPSCsnoIncreased Iysosomal activity7
HD 50150Adult onset HDunknown (father)OSKM (retrovirus)AstrocytenoNeural differentiation normal, Vacuolation in astrocyte12
HD109-11109Juvenile HDunknown (daughter)OSKM (retrovirus)AstrocytenoSimilar to HD 50, more vacuolation in astrocyte12
HD 72172Juvenile HD20 yearsOSKM (retrovirus)NPCsyesElevated caspase activity; more vulnerable to cell death6,8,9
HD 60360Adult onset HD29 years2 lines:OSKMNL (lentivirus) 1 line: OSKM (episomal)NPCs, neuronsnoAltered cell adhesion, energetics, and electrophysiology; Increased cell death in long time neural differentiation5
HD109-21109Juvenile HD9 yearsOSKMNL (lentivirus)NPCs, neuronsnoSimilar to HD 60; higher risk to cell death in response to BDNF withdrawal5
HD1804180Juvenile HD6 years3 lines:OSKMNL (lentivirus) 1 line: OSKM (episomal)NPCs, neuronsnoSimilar to HD 60 and 109; Increased vulnerable to stress and toxicity5
Open in a separate windowHD, Huntington''s Disease; iPSC, induced pluripotent stem cell; NPC, neural progenitor cell; O, Oct4; S, Sox2; K, Klf4; M, Myc; N, Nanog; L, Lin28.Meanwhile, using a homologous recombination-based gene targeting strategy, An et al.6 reported on the successful correction of the CAG-repeat-expanded HTT allele in HD patient iPSCs. These corrected iPSCs shared the same genetic background as the disease iPSCs, thereby serving as non-biased controls for their uncorrected counterparts. By comparing gene expression profiles of corrected iPSCs versus disease iPSCs, An et al. found that the alterations of cadherin, TGF-β, and caspase-related pathways in HD were rescued in the non-expanded iPSCs. The authors further demonstrated that gene correction in HD iPSCs reversed disease phenotypes such as susceptibility to cell death and altered mitochondrial bioenergetics in NSCs. More importantly, when transplanted into a mouse model of HD, the corrected HD iPSC-derived NSCs could survive and differentiate into GABAergic neurons and DARPP-32-positive neurons in vivo.Taken together, these two studies present very significant advances for iPSC-based disease modeling of HD and provide a potential donor source for cell replacement therapy. Though exciting indeed, several important challenges remain unsolved.First, complete recapitulation of neuropathology phenotypes in the iPSC-based models in vitro remains a challenge in the field. As a neurodegenerative disease, pathologic development of HD usually takes several decades and may be influenced by several external factors. In the HD iPSC-based model, the derivation method, clonal discrepancy as well as the culture conditions may affect the manifestation of phenotypes. Indeed, in previously reported HD iPSC lines, only slight increases in caspase and lysosomal activity were observed7,8,9. Although in both reports of HD iPSCs, significant phenotypes in electrophysiology, energy metabolism and cell death were recorded, other typical HD-associated phenotypes such as oligomeric mutant HTT aggregation, formation of nuclear inclusions and preferential striatal degeneration were not observed.Second, it is still an open question whether neural cells derived from gene-corrected iPSCs are fully functional, that is, whether they may restore physiological functions after cell replacement therapy. Ma et al.10 have recently reported on a protocol to differentiate striatal projection neurons from human embryonic stem cells with a high efficiency. After transplantation, these cells survived, reconnected striatal circuitry, and restored motor function in a striatal neurodegenerative mouse model. In spite of these encouraging first attempts, further improvements of the methodology for the directed cell differentiation in vitro and cell transplantation in vivo are still needed.Third, HTT protein is ubiquitously expressed and functional in different tissue. It has been hypothesized that HD may also develop in a non-autonomous manner11. The current studies mainly focused on the phenotypes of HD iPSC-derived neurons. However, supporting cells such as astrocytes might also play direct or indirect roles in HD progression. Indeed, a vacuolation phenotype has been observed in HD iPSC-derived astrocytes12. Therefore, it will be interesting to expand the HD iPSC platform into other cell types with the goal to extend and uncover the various ethiopathological factors involved in HD.Finally, human iPSC models of monogenic disorders in general possess great potential for the mechanistic study of the disease. However, as is the case with many neuropsychiatric disorders, HD establishment and progression is linked to different genetic and epigenetic factors, including environmental change-induced epigenetic modification, multiple mutations, and genetic alternation in non-coding regions. To this end, although the successful generation of HD iPSCs as well as targeted gene correction would greatly facilitate the study of HD, a comprehensive understanding of HD pathogenesis will need to be achieved before trying to translate the recent results into the clinic.In summary, despite all of these open questions, the recent studies have uncovered the unlimited potential of iPSCs for modeling HD in vitro. These studies will promote and enhance HD research in various areas, including elucidation of HD cellular pathogenesis, development of HD-specific biomarkers, screening for small therapeutic molecules, and manipulation of HD iPSCs for stem cell replacement therapy, which together may ultimately fulfill the promise of using iPSCs as a tool for regenerative medicine and drug discovery for HD in the near future.  相似文献   

15.
Bacterial regeneration of ammonium and phosphate as affected by the carbon:nitrogen:phosphorus ratio of organic substrates   总被引：10，自引：2，他引：8  
Yasuhiko Tezuka 《Microbial ecology》1990,19(3):227-238
The effect of carbonnitrogenphosphorus (CNP) ratio of organic substrates on the regeneration of ammonium and phosphate was investigated by growing natural assemblages of freshwater bacteria in mineral media supplemented with the simple organic C, N, and P sources (glucose, asparagine, and sodium glycerophosphate, respectively) to give 25 different substrate CNP ratios. Both ammonium and phosphate were regenerated when CN and NP atomic ratios of organic substrates were 101 and 161, respectively. Only ammonium was regenerated when CN and NP ratios were 101 and 10–201, respectively. On the other hand, neither ammonium nor phosphate was regenerated when CN and NP ratios were 151 and 51, respectively. In no case was phosphate alone regenerated. As bacteria were able to alter widely the CNP ratio of their biomass, the growth yield of bacteria appeared primarily dependent on the substrate carbon concentration, irrespective of a wide variation in the substrate CNP ratio.  相似文献   

16.
Differential Phenotypic Diversity among Epidemic-Spanning Salmonella enterica Serovar Enteritidis Isolates from Humans or Animals     
Lucía Yim  Laura Betancor  Arací Martinez  Gerardo Giossa  Clare Bryant  Duncan Maskell  Jose A. Chabalgoity 《Applied and environmental microbiology》2010,76(20):6812-6820
  相似文献   

17.
Genomic and Biochemical Analysis of N Glycosylation in the Mushroom-Forming Basidiomycete Schizophyllum commune     
Elsa Berends  Robin A. Ohm  Jan F. de Jong  Gerard Rouwendal  Han A. B. W?sten  Luis G. Lugones  Dirk Bosch 《Applied and environmental microbiology》2009,75(13):4648-4652
  相似文献   

18.
Transcriptional Repression by the SMRT-mSin3 Corepressor: Multiple Interactions,Multiple Mechanisms,and a Potential Role for TFIIB          下载免费PDF全文
Chi-Wai Wong  Martin L. Privalsky 《Molecular and cellular biology》1998,18(9):5500
  相似文献   

19.
Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments     
Nidiane D. R. Prado  Soraya S. Pereira  Michele P. da Silva  Michelle S. S. Morais  Anderson M. Kayano  Leandro S. Moreira-Dill  Marcos B. Luiz  Fernando B. Zanchi  André L. Fuly  Maribel E. F. Huacca  Cleberson F. Fernandes  Leonardo A. Calderon  Juliana P. Zuliani  Luiz H. Pereira da Silva  Andreimar M. Soares  Rodrigo G. Stabeli  Carla F. C. Fernandes 《PloS one》2016,11(3)
Antivenoms, produced using animal hyperimmune plasma, remains the standard therapy for snakebites. Although effective against systemic damages, conventional antivenoms have limited efficacy against local tissue damage. Additionally, the hypersensitivity reactions, often elicited by antivenoms, the high costs for animal maintenance, the difficulty of producing homogeneous lots, and the instability of biological products instigate the search for innovative products for antivenom therapy. In this study, camelid antibody fragments (VHH) with specificity to Bothropstoxin I and II (BthTX-I and BthTX-II), two myotoxic phospholipases from Bothrops jararacussu venom, were selected from an immune VHH phage display library. After biopanning, 28 and 6 clones recognized BthTX-I and BthTX-II by ELISA, respectively. Complementarity determining regions (CDRs) and immunoglobulin frameworks (FRs) of 13 VHH-deduced amino acid sequences were identified, as well as the camelid hallmark amino acid substitutions in FR2. Three VHH clones (KF498607, KF498608, and KC329718) were capable of recognizing BthTX-I by Western blot and showed affinity constants in the nanomolar range against both toxins. VHHs inhibited the BthTX-II phospholipase A2 activity, and when tested for cross-reactivity, presented specificity to the Bothrops genus in ELISA. Furthermore, two clones (KC329718 and KF498607) neutralized the myotoxic effects induced by B. jararacussu venom, BthTX-I, BthTX-II, and by a myotoxin from Bothrops brazili venom (MTX-I) in mice. Molecular docking revealed that VHH CDRs are expected to bind the C-terminal of both toxins, essential for myotoxic activity, and to epitopes in the BthTX-II enzymatic cleft. Identified VHHs could be a biotechnological tool to improve the treatment for snake envenomation, an important and neglected world public health problem.  相似文献   

20.
Cross-Subtype Neutralization Sensitivity despite Monoclonal Antibody Resistance among Early Subtype A,C, and D Envelope Variants of Human Immunodeficiency Virus Type 1     
Catherine A. Blish  Zahra Jalalian-Lechak  Stephanie Rainwater  Minh-An Nguyen  Ozge C. Dogan  Julie Overbaugh 《Journal of virology》2009,83(15):7783-7788
The human immunodeficiency virus type 1 (HIV-1) variants that are transmitted to newly infected individuals are the primary targets of interventions, such as vaccines and microbicides, aimed at preventing new infections. Newly acquired subtype A, B, and C variants have been the focus of neutralization studies, although many of these viruses, particularly of subtypes A and B, represent viruses circulating more than a decade ago. In order to better represent the global diversity of transmitted HIV-1 variants, an additional 31 sexually transmitted Kenyan HIV-1 env genes, representing several recent infections with subtype A, as well as subtypes A/D, C, and D, were cloned, and their neutralization profiles were characterized. Most env variants were resistant to neutralization by the monoclonal antibodies (MAbs) b12, 4E10, 2F5, and 2G12, suggesting that targeting the epitopes of these MAbs may not be effective against variants that are spreading in areas of endemicity. However, significant cross-subtype neutralization by plasma was observed, indicating that there may be other epitopes, not yet defined by the limited available MAbs, which could be recognized more broadly.Most effective viral vaccines are thought to provide protection primarily by stimulating neutralizing antibodies (NAbs) to clear cell-free virus (25, 27). Because protection by NAbs requires recognition of common viral epitopes, the extreme genetic diversity of human immunodeficiency virus type 1 (HIV-1) presents a particular challenge to NAb-based vaccine approaches. Therefore, a critical starting point for studies of immune-mediated protection against HIV-1 is a collection of newly transmitted HIV-1 variants, particularly from areas of endemicity, such as sub-Saharan Africa, in order to determine whether vaccines are appropriately targeted to common epitopes from these relevant transmitted strains.During HIV-1 transmission, a bottleneck allows only one or a few variants to be transmitted to a newly infected individual (6, 9, 16, 29, 34, 37, 39), and the sensitivity of these early transmitted strains to antibody-mediated neutralization is therefore of particular interest. Newly transmitted HIV-1 variants have demonstrated significant heterogeneity in their neutralization phenotypes both within and between subtypes (2, 3, 6-8, 11, 13-15, 22, 30, 32, 36). Panels of sexually transmitted HIV-1 envelope variants (based on the envelope gene, env) have been characterized, including subtype B variants from North America, Trinidad, and Europe, subtype C variants from South Africa and Zambia, and subtype A variants from Kenya collected between 1994 and 1996 (2, 14, 15). Here, we characterize an additional 31 envelope variants from 14 subjects with sexually transmitted HIV-1 who were infected in Kenya, where subtypes A, C, and D circulate, between 1993 and 2005 (24, 31).The env genes were cloned from samples drawn 14 to 391 (median, 65) days postinfection from individuals enrolled in a prospective cohort of high-risk women in Mombasa, Kenya (19-21). Demographic characteristics of the subjects are summarized in Table ​Table1;1; the timing of first infection was determined by both HIV-1 serology and HIV RNA testing as described previously (12). All of the subjects were presumably infected by male-to-female transmission and displayed a range of plasma viral loads at the time of env gene cloning (Table ​(Table1).1). For most individuals, full-length env genes were cloned from uncultured peripheral blood mononuclear cell (PBMC) DNA, though for two individuals, clones were obtained from DNA following short-term coculture with donor PBMCs (Table ​(Table1).1). env genes were cloned by single-copy nested PCR with primers and PCR conditions as described previously (4, 17). We tested env genes for their ability to mediate infection by transfecting env plasmid DNA into 293T cells along with an env-deficient HIV-1 subtype A proviral plasmid, Q23Δenv, to make pseudoviral particles (17). More than 80 env clones were obtained from 16 subjects; less than one-half were functional on the basis of the infectivity of pseudoviral particles in a single-round infection of TZM-bl cells (AIDS Research and Reference Reagent Program, National Institutes of Health), as observed previously for env genes cloned from proviral sequences (17); a lower fraction of functional env genes have been reported from plasma (18). We focused on the proviral sequences here because they presumably best represent the sequence closest to that of the transmitted strains. The 31 functional env variants are described in Table ​Table11.

TABLE 1.

Demographic characteristics, diversities, gp120 variable-region lengths, numbers of PNGS, and accession numbers of cloned env variants
SubjectVirus subtypeSample date (mo/day/yr)dpiaPlasma VLbSourcecIndividual env clonePairwise difference (%)dVariable-loop length (aa)
No. of PNGS
GenBank accession no.
V1/V2V3V4V5gp120gp41gp41 ecto
QB726A04/16/967061,940ucPBMCQB726.70M.ENV.B30.16633536102244FJ866111
QB726.70M.ENV.C4633536102244FJ866112
QF495A05/16/0623217,050ucPBMCQF495.23M.ENV.A10.121073537113044FJ866113
QF495.23M.ENV.A31073537113044FJ866114
QF495.23M.ENV.B21133537113144FJ866115
QF495.23M.ENV.D11133537113144FJ866116
QG984A07/12/042130,300ucPBMCQG984.21M.ENV.A3NA693436112433FJ866117
QH209A10/13/051428,600ucPBMCQH209.14M.ENV.A2NA723529112444FJ866118
QH343A09/08/052140,750,000ucPBMCQH343.21M.ENV.A100.19773532152644FJ866119
QH343.21M.ENV.B5773532152644FJ866120
QH359A10/05/052132,120ucPBMCQH359.21M.ENV.C11.4843536102944FJ866121
QH359.21M.ENV.D1733535102644FJ866122
QH359.21M.ENV.E2723540132844FJ866123
QA790eA/D06/10/9620448,100ccPBMCQA790.204I.ENV.A40.36773533112544FJ866124
QA790.204I.ENV.C1773533112644FJ866125
QA790.204I.ENV.C8773533112444FJ866126
QA790.204I.ENV.E2773533112544FJ866127
QG393A2/D06/23/046017,360ucPBMCQG393.60M.ENV.A10.7603431102455FJ866128
QG393.60M.ENV.B7573431102455FJ866129
QG393.60M.ENV.B8573431102455FJ866130
QB099eC02/10/9539127,280ucPBMCQB099.391M.ENV.B10.43653529102544FJ866131
QB099.391M.ENV.C8653529102544FJ866132
QC406C07/08/9770692,320ucPBMCQC406.70M.ENV.F3NA643520112254FJ866133
QA013D10/11/95701,527,700ccPBMCQA013.70I.ENV.H10.16603429122544FJ866134
QA013.70I.ENV.M12603429122544FJ866135
QA465D08/19/935937,750ucPBMCQA465.59M.ENV.A10.24653530112844FJ866136
QA465.59M.ENV.D1653530112744FJ866137
QB857D10/16/9711014,640ucPBMCQB857.23I.ENV.B3NA683432112654FJ866138
QD435D04/06/9910017,470ucPBMCQD435.100M.ENV.A40.88693429122654FJ866139
QD435.100M.ENV.B5673429112454FJ866140
QD435.100M.ENV.E1693429122654FJ866141
Open in a separate windowadpi, days postinfection as defined by RNA testing (12).bVL, viral load on the sample date in which env genes were cloned.cucPBMC, uncultured PBMCs; ccPBMC, cocultured PBMCs.dAverage pairwise distance between the full-length env variants from a given subject. NA, not applicable because there was only one variant available from the subject.eenv variants from these two subjects were cloned from >6 months postinfection, as noted, and should not be considered true early env variants.The full-length, functional env genes were sequenced and aligned to generate a maximum likelihood phylogenetic tree with reference sequences from the Los Alamos National Laboratory HIV database, as described previously (26). Viral env clones from the same subject clustered together, and a wide spectrum of genetic diversity was observed overall (Fig. ​(Fig.1).1). Some women, such as subject QF495, were infected with a relatively homogeneous viral population, with average pairwise differences of only 0.12% between env variants (Table ​(Table11 and Fig. ​Fig.1).1). However, as observed previously in this cohort (16, 28, 29, 33-35), other individuals, such as subjects QH359 and QD435, were infected with more heterogeneous viral populations with average pairwise differences of 1.4% and 0.88% between variants, respectively (Table ​(Table11 and Fig. ​Fig.1).1). env genes from subtypes A (13 variants), C (3 variants), and D (8 variants), as well as A/D recombinants (4 variants) and A2/D recombinants (3 variants), were represented (Fig. ​(Fig.1).1). The viral subtypes were confirmed using the NCBI genotyping database (http://www.ncbi.nlm.nih.gov/).Open in a separate windowFIG. 1.Maximum likelihood phylogenetic tree of full-length sequences from early subtype A, C, D, and A/D recombinant env variants in Kenya. The 31 novel env clones from Kenyan early infections and reference sequences for subtypes A, B, C, D, and K from the Los Alamos HIV database (http://www.hiv.lanl.gov/content/index) are displayed. The phylogenetic tree was rooted with subtype K env sequences. Values at nodes indicate the percentage of bootstraps in which the cluster the right was found; only values of 70% or greater are shown.The deduced amino acid sequences revealed that all functional variants had an uninterrupted open reading frame in env except for variant QB099.391I.ENV.C8, which had a frameshift mutation within the cytoplasmic tail of gp41. There was significant heterogeneity in the length of the protein variable loops, particularly V1/V2, which ranged from 57 amino acids (aa) to 113 aa (Table ​(Table1).1). The V3, V4, and V5 loops also varied in length, though less dramatically (Table ​(Table1).1). Variants from the same subject were generally similar in their variable-loop lengths. Moderate variation was also observed in the number and position of potential N-linked glycosylation sites (PNGS) (Table ​(Table11).Previous analyses indicated that early subtype C env proteins had shorter variable loops than did early subtype B env proteins (13), suggesting that there are different env protein features between subtypes. Thus, to compare variable-loop lengths and the numbers of PNGS between subtypes using this expanded group of early env variants, we evaluated the 31 newly cloned variants plus an additional 15 subtype A variants (2), 19 subtype B variants (14), and 18 subtype C variants (15) from other early virus panels. In order to avoid bias, when more than one env variant was available from a subject, the average loop length or PNGS number for that subject''s env proteins was used. We did not observe significant differences in V1/V2 length, V5 length, or the numbers of PNGS between subtypes by the Kruskal-Wallis equality-of-populations rank test (Table ​(Table2)2) . However, there were significant differences between the V3 and V4 loop lengths of the subtypes after adjusting for multiple comparisons (Table ​(Table2).2). The differences in V3 length appeared to be a result of shorter V3 loops in subtype D env proteins than in early subtype B (P = 0.006) or C (P < 0.001) env proteins (Table ​(Table2).2). The differences in V4 length were caused by shorter V4 loops in subtype C env proteins in comparison to both subtype A and B env proteins (P < 0.001; Table ​Table22).

TABLE 2.

Summary of variable-loop lengths and the numbers of PNGS in gp120 and gp41 within early HIV-1 env variantsa
ParameterMedian value (25th percentile, 75th percentile) for subtype:
Kruskal- Wallis P valuebWilcoxon rank sum P values for individual comparisonsc
A (n = 11)B (n = 19)C (n = 20)D (n = 4)A vs. BA vs. CA vs. DB vs. CB vs. DC vs. D
Length
    V1/V270.3 (62, 76)70 (66, 70)65 (62, 76)66.5 (62, 69)0.210.7300.2820.2150.0510.1130.846
    V335 (34, 35)35 (35, 35)35 (34, 35)34 (34, 35)0.0010.2400.0160.1070.1410.006<0.001
    V432 (30, 36)33 (31, 34)26.5 (22, 29)29.5 (29, 31)0.00010.880<0.0010.148<0.0010.0230.056
    V511 (11, 11)10 (9, 11)10 (9, 11)11.5 (11, 12)0.0300.0960.0150.1840.6770.0990.021
No. of PNGS in:
    gp12024 (23, 28)25 (24, 26)24 (23, 25)26 (26, 27)0.200.6800.6920.2650.1460.1860.042
    gp414 (4, 5)5 (4, 5)5 (4, 5)4.5 (4, 5)0.200.0300.1790.4700.4100.4080.799
    gp41ecto4 (4, 4)4 (4, 4)4 (4, 5)4 (4, 4)0.0440.1070.0250.5500.0880.5070.201
Open in a separate windowaVariable-loop lengths and the numbers of PNGS in gp120 and gp41 within early HIV-1 env variants from subtypes A, B, C, and D characterized here and previously (2, 14, 15). n, number of samples.bKruskal-Wallis equality-of-populations rank test (based on multiple comparisons; P values of <0.007 were considered significant; significant values are presented in boldface).cWilcoxon rank sum test (based on multiple comparisons; P values of <0.008 were considered significant; significant values are presented in boldface).We then assessed the neutralization sensitivity of the pseudoviruses to antibodies in plasma from HIV-1-infected individuals and to HIV-1-specific MAbs by using the TZM-bl neutralization assay as described previously (2, 23, 38). Median inhibitory concentrations (IC50s) were defined as the reciprocal dilution of plasma or concentration of MAb that resulted in 50% inhibition of infection (2, 38). The Kenya pool was derived by pooling plasma collected between 1998 and 2000 from 30 HIV-1-infected individuals in Mombasa, Kenya, and the other three pools were derived by pooling plasma collected between 1993 and 1997 from 10 individuals from Nairobi, Kenya, and with an infection with a known subtype (A, C, or D) of HIV-1 as described previously (2).The env variants demonstrated a range of neutralization sensitivities to plasma samples, from neutralization resistant (defined as <50% neutralization with a 1:50 dilution of plasma) to neutralization sensitive with an IC50 of 333 (Fig. ​(Fig.2).2). Some clones, such as QF495.23M.ENV.A1, were relatively sensitive to all the plasma pools, with IC50s from 100 to 333, whereas other clones, such as QH343.21M.ENV.A10, were relatively resistant to these plasma pools, with IC50s from <50 to 85 (Fig. ​(Fig.2).2). The plasma pools did differ in their neutralization potencies. The Kenya pool, with a median IC50 of <50 across all viruses tested, was significantly less likely to neutralize these transmitted variants than were the subtype A, C, and D plasma pools, which had median IC50s of 110, 105, and 123, respectively (P values of <0.0001, 0.0001, and 0.001, respectively, by paired t test on log-transformed IC50s). The basis for these differences in neutralizing activity is not clear, although the location, timing, and level of immunodeficiency at the time of sample collection could have contributed to the differences in NAb levels between the pools.Open in a separate windowFIG. 2.Neutralization sensitivity of early subtype A, C, D, and A/D recombinant env variants to plasma samples and MAbs in relation to the sequences of the MAb binding sites. The env used to generate the pseudovirus tested is shown at the left, and the plasma pool or MAb tested is indicated at the top. The IC50s of each plasma sample or MAb against each viral pseudotype is shown, with darker shading indicating more potent neutralization, as defined at the bottom of the figure. Gray boxes indicate that <50% neutralization was observed at the highest dilution of plasma or concentration of MAb tested. Each IC50 shown is an average of the results from two independent neutralization assays, using pseudovirus generated in independent transfection experiments. The median IC50s from the 31 variants are shown at the bottom. Neutralization of the pseudovirus derived from the subtype B variant SF162 is shown as a control, and neutralizations of murine leukemia virus (MLV) and simian immunodeficiency virus clone 8 (SIV) are shown as negative controls. In the panels on the right, the sequences for the MAbs 2G12, 2F5, and 4E10 are displayed. For 2G12, the amino acid numbers for the five PNGS that are important for 2G12 binding are shown for each virus tested. A plus sign indicates that the PNGS at that site in the envelope sequence was preserved, and a minus sign indicates that the PNGS was deleted. A shift in the PNGS position is indicated by the amino acid position to which the PNGS shifted. All sequences were numbered relative to the HXB2 sequence. The two rightmost panels show data for the canonical 2F5 and 4E10 epitopes, with a period indicating that the amino acid is preserved.The env variants were significantly more susceptible to their subtype-matched plasma pool, with a higher mean IC50 for subtype-matched plasma samples than for unmatched plasma samples (138 versus 108, P = 0.0081, paired t test). However, a significant amount of cross-subtype neutralization was observed, as every env variant that was susceptible to the subtype-matched plasma pool was also susceptible to at least one of the other plasma pools (Fig. ​(Fig.2).2). Thus, although potency was enhanced when the plasma antibodies were produced in response to infection with the same subtype of HIV-1, there were shared neutralization determinants between subtypes, as has been observed previously (reviewed in reference 3).To identify potential correlates of neutralization sensitivity to the antibodies within these plasma pools, we included these 31 env variants and an additional 15 subtype A env variants we previously characterized from the same cohort with the same plasma pools (2). We did not observe a change in neutralization sensitivity during the evolution of the HIV-1 epidemic in Kenya, as no correlation was observed between neutralization sensitivity and the calendar date from which the env variants were isolated. In addition, no correlation was observed between the neutralization sensitivity of a variant to the plasma pools and the duration of estimated infection within that individual. Finally, there was no significant correlation between the neutralization sensitivity and variable-loop length or the number of PNGS. Thus, although changes in the variable-loop length or number of PNGS may alter the exposure of epitopes within the HIV-1 env protein, these changes do not appear to be the primary determinant of neutralization sensitivity.Despite relatively universal sensitivity to at least one of the pooled plasma samples, these transmitted Kenyan env variants were generally resistant to the MAbs 2G12 (provided by Hermann Katinger, Polymun Scientific) and b12 (provided by Dennis Burton, The Scripps Research Institute), as well as 2F5 and 4E10 (obtained from the AIDS Research and Reference Reagent Program, National Institutes of Health) (Fig. ​(Fig.2),2), though these MAbs neutralized the subtype B env variant SF162, with IC50s similar to those reported previously (1). Subtype D strains were the most susceptible to MAbs, with 4/8 variants neutralized with <20 μg/ml of 2F5 and 2/8 neutralized with <20 μg/ml of the other MAbs. This could reflect the fact that subtype D variants are more closely related to subtype B strains (Fig. ​(Fig.1)1) (see reference 10), and these MAbs were all derived from subtype B-infected individuals.Among all 31 variants, 2F5 was the most broadly neutralizing, with 15/31 variants from 8/14 subjects neutralized with <20 μg/ml of this MAb. Some 2F5-resistant env variants, such as QH209.14M.ENV.A2 and QB857.110I.ENV.B3, had mutations in the canonical 2F5 binding epitopes, though other 2F5-resistant env variants such as QF495.23M.ENV.A3 and QA790.204I.ENV.A4 maintained the canonical 2F5 epitope. The results with the MAb 4E10 were similar; 4E10 neutralized only seven variants from 4 of the 14 subjects, and the presence of mutations in the 4E10 epitope, which were common, did not predict neutralization sensitivity (Fig. ​(Fig.2).2). For instance, the env variants QH343.21M.ENV.A10 and QH343.21M.ENV.B5 contained identical N671S and D674S mutations and QH343.21M.ENV.B5 was highly sensitive to 4E10, while QH343.21M.ENV.A10 was resistant (Fig. ​(Fig.2).2). Thus, for the 2F5 and 4E10 epitopes, the presumed epitopes appear to be shielded in a subset of these early non-subtype B env variants, as has been previously observed (Fig. ​(Fig.2)2) (1, 2, 5, 14).The MAb b12 neutralized only two variants from two subtype D-infected individuals, with no neutralization of the subtype A, C, and A/D recombinant pseudoviruses. Only four variants from two subjects were neutralized by 2G12 at <20 μg/ml, and these were the only variants that maintained all five of the PNGS within the 2G12 epitope (Fig. ​(Fig.2).2). Overall, the median IC50 of all the MAbs against these transmitted variants was >20 μg/ml. None of the variants was susceptible to all four MAbs (Fig. ​(Fig.2),2), unlike many of the early subtype B env variants characterized previously (14).In summary, these newly characterized HIV-1 env clones represent a range of neutralization sensitivities and can be used to supplement existing panels of transmitted variants, in particular, adding the first subtype D and A/D recombinant variants. Some differences between subtypes in env structure following transmission were noted, though these differences did not correlate with neutralization sensitivity. Although the significant levels of cross-subtype neutralization sensitivity observed with plasma samples indicate that some neutralization determinants were shared across subtypes, the epitopes for the MAbs b12, 2G12, 2F5, and 4E10 did not appear to be among the shared determinants. Thus, despite the fact that significant attention has focused on using vaccination to develop antibodies that resemble these MAbs in their specificity, such antibodies may not neutralize the transmitted strains that are causing most new infections worldwide. These data therefore stress the importance of evaluating transmitted variants in endemic areas when designing immunogens and evaluating vaccine and microbicide strategies.  相似文献   

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