Candidatus Liberibacter asiaticus (CLas), the causal agent of the citrus disease Huanglongbing (HLB), and its insect vector, the Asian citrus psyllid , have been devastating the Florida citrus industry. To restore the competitive production presence of Florida in the worldwide citrus market, effective and sustainable control of HLB and the ACP needs to be identified. As alternatives for resistance-inducing insecticides, viruses are currently being considered for biological control of the ACP. To identify possible biological control candidates, we conducted one of the most comprehensive surveys of natural ACP populations in major citrus production regions spanning 21 counties in Florida. By optimizing PCRs and RT-PCRs, we were able to successfully detect and monitor the prevalence of five previously identified ACP-associated RNA and DNA viruses throughout Florida citrus groves, which include: Diaphorina citri-associated C virus (DcACV), Diaphorina citri flavi-like virus (DcFLV), Diaphorina citri densovirus (DcDNV), Diaphorina citri reovirus (DcRV), and Diaphorina citri picorna-like virus (DcPLV). Adult and nymph ACP populations from 21 of Florida's major citrus-producing counties were collected each month during approximately 18 consecutive months. RNA extracts used for these viral screens were also regionally combined and subjected to High Throughput Sequencing (HTS) to reveal a more comprehensive picture of known and unknown viruses in Florida ACP populations. We discovered that DcACV was the most prevalent ACP-associated virus throughout nymph and adult ACP populations in Florida, detected in more than 60% of all samples tested, followed by DcPLV and DcFLV. HTS allowed us to identify a novel ACP-associated reo-like virus and a picorna-like virus. The putative reo-like virus, tentatively named Diaphorina citri cimodo-like virus, was later surveyed and detected back in seasonal adult and nymph ACP samples collected in Florida during this study. HTS generated data also revealed that the most abundant virus in Florida ACP populations was Citrus tristeza virus (CTV), which is not an ACP-associated virus, suggesting persistent presence of CTV infection in citrus throughout Florida groves. Collectively, information obtained from our study may be able to help guide the direction of biotechnological pest control efforts involving a number of viruses that were detected for the first time in Florida ACP populations, including two newly identified ACP-associated viruses.The plant pathogenic bacterium  Candidatus Liberibacter asiaticus (CLas), the causal agent of the citrus disease Huanglongbing (HLB), continues to endanger the worldwide citrus industry, with the most severe losses occurring in Florida , has been proven to be the primary means of dispersal of this disease throughout citrus groves in Florida , which has been documented as an effective way to deliver RNA interference (RNAi) into both plants and insects, is a molecular strategy for the biological control of the ACP and eventual disruption of the HLB disease cycle (Citrus tristeza virus (CTV), is currently being used as a viral vector to deliver this RNAi technology into the citrus phloem and then into the ACPs that feed on citrus  in the ACP vector itself represent our strategy to control the disease without relying solely on chemical applications. The necessity of a precise and specific biological control for Florida ACP populations initiated this study, with hopes to provide additional molecular tools. In order to decrease or eliminate major dependence on broad-spectrum chemical controls, this study aimed to investigate the Florida ACP virome for identifying an ACP-associated virus with the potential to be manipulated into a biocontrol agent against the HLB insect vector. The endemic establishment of both HLB and the ACP in Florida has greatly increased the need for alternative control options.As a viable alternative to chemical controls, development of knowledge-based and efficient biological control strategies to disrupt D. citri, such as a picorna-like virus, a reovirus, a densovirus, a bunyavirus, and an unclassified (+) ssRNA virus  (unpublished data) lead us to our current strategy for identifying and detecting ACP-associated viruses, especially in Florida ACP populations. Additionally, CLas survival, reproduction, and dissemination has been observed to occur principally in the gut of the ACP, which has influenced supplementary targeted approaches for potentially inhibiting bacterial persistence in this organ  technologies. The first objective will provide foundational knowledge of the known viruses that might already be present in Florida ACP populations, with regards to their spatial and temporal characteristics throughout citrus-producing counties. The second objective will provide a more thorough analysis of the Florida ACP virome, including the detection of novel and other known viruses. By analyzing ACPs from around the world, Adult and nymph ACPs were collected directly from citrus trees in commercial groves in 21 of Florida's major citrus-producing counties around south-central Florida every month over a period of approximately eighteen months (August 2017 through December 2018). One grove, ranging from 500 to 2,000 acres (~200 to 800 hectare), was sampled per county and samples were taken randomly from multiple trees in one block of each grove usually around the perimeter of the block, where the observation of ACP incidence has been the greatest . Each ciYoung citrus shoots with adult or nymph ACPs were carefully detached from trees and immediately submerged into 50 ml Falcon tubes containing 100% ethanol, killing the ACPs, and the Falcon tubes were kept closed. Since HLB and ACPs are currently widespread in Florida, traveling throughout Florida with (dead) ACPs was not a concern. The number of ACPs collected varied by grove; however, we aimed to collect a minimum of 25 adults and 25 nymph ACPs from each grove every month. There were times in which lower numbers were collected due to inhospitable conditions, such as cooler or rainy weather, or increased pesticide applications. ACPs collected from these 21 groves were brought to the laboratory at the Southwest Florida Research and Education Center (SWFREC) in Immokalee FL, where adult and nymph ACPs were separated out from plant parts, counted with the aid of a stereo microscope , and stored separately in smaller vials containing 100% ethanol at -20°C until needed. Total ACP counts per grove site (representing one county) were then calculated and combined into six regions for data analysis  of the psyllid population in each county and each month. Preliminary tests (data not shown) and previous research together determined that five insects of this size (2–5 mm) was sufficient to yield enough total RNA for RT-PCR screenings (Diaphorina citri (Dc) wingless gene as an internal control   were mixed and incubated at 65°C for 5 min followed by incubation on ice for 5 min to begin complementary DNA (cDNA) synthesis. One microliter of Superscript II (200 u) reverse transcriptase , 1 μl of dNTPs (10 mM), 2 μl of DTT (0.1 mM) , 4 μl of buffer (5X)  and 4.5 μl of RNAse-free HDiaphorina citri-associated C virus (DcACV), Diaphorina citri flavi-like virus (DcFLV), Diaphorina citri picorna-like virus (DcPLV), Diaphorina citri reovirus (DcRV), and Diaphorina citri densovirus (DcDNV), respectively (Dc) wingless gene, was also used in order to verify the integrity of the RNA for each sample. The PCR primers used for each virus, along with the target gene and the length of the amplicon, are listed in Dc] wingless gene) for 20 s and 72°C for 2 min, with final extension at 72°C for 10 min. PCR products were separated on 1% agarose gels and visualized by staining with Apex™ Safe DNA Stain . Randomly selected amplicons with expected size generated from genomic DNA (DcDNV) and cDNA , and from corresponding positive control plasmids  were purified using the DNA Clean and Concentrate kit . The purified DNA products were sent for Sanger sequencing  and generated sequences were subjected to the National Center for Biotechnology Information Basic Local Alignment Search Tool (NCBI BLAST) program to validate the target amplicons' identity.The cDNA and total RNA from all samples were used to screen all monthly-collected county samples for four previously identified ACP-associated RNA viruses and one DNA virus: ectively . These fectively . An inteThe six larger Florida regions (Regions A–F), as depicted in All six RNA samples were sent to Foundation Plant Services at UC Davis, CA, USA. Here, separate aliquots of the six samples of total RNAs were prepared and each subjected to ribosomal RNA depletion and subsequent cDNA library construction using a TruSeq Stranded Total RNA with Ribo-Zero Plant kit . All six samples were then sequenced on the Illumina NextSeq 500 platform as previously described .de novo assembled into contiguous consensus sequences (contigs) with size lengths of at least 200 base pairs by CLC Bio Genomic Workstation . These contigs were then annotated by a multistep process for generation of candidate viral sequences as previously described (https://www.ncbi.nlm.nih.gov/genome/viruses/)  . It was Based on the assembled list of contigs compiled from HTS results, RT-PCR primers were designed to target the 5' and 3' ends of a novel viral sequence detected in HTS. The sequences of the designed primers are shown in P < 0.05.For statistical analysis of ACP-associated viruses, the results for the individual county-collected samples were combined into the same six Florida regions that were established for HTS analysis  adults and nymphs from each citrus grove were collected monthly  were detected in all regions sampled whereas DcRV was detected in only Region A and D (The DcFLV was found to exist at very close incidence levels to DcPLV (in 19% of the samples) and was detected in both adult and nymph ACPs in all regional Florida samples, though at much lower incidence levels than DcACV; however, it was found to be the third most prevalent virus (P < 0.05). For example, Region A  wingless gene (data not shown). The ACP wingless gene was used for the confirmation of total RNA and cDNA integrities during this study. The detection of the wingless gene added further confidence in negative samples of the ACP-associated viral survey results.The cDNA samples generated from RNA extracts during the study always tested positive for the ACP internal control, the ACP (Diaphorina citri cimodo-like virus (DcCLV), was detected in both adult and nymph ACP populations. It was found in all of the six Florida Regions; however, it excluded most of the seasons in Region B, D, and E (HTS yielded between 24 and 35 million raw reads per cDNA library (data not shown). Subsequent analysis identified sequences of several diverse viruses in all ACP samples. As expected, some of these viral sequences shared high similarities with sequences of known ACP-associated viruses, including DcACV and DcFLV. However, there was also a substantial number of sequences sharing similarities with novel viruses that were not known to be present in the ACP (data not shown). For example, a novel virus sequence (~ 4.1 kb in length) with very low similarity (< 50%) to the RNA-dependent RNA polymerase (RdRp) gene of another reovirus from an insect in Africa  was discCitrus tristeza virus (CTV) sRNA sequences in all Florida regional ACP samples compared to any other known virus. More than 60% of all sRNA reads in each regional sample were matched to the CTV genome, with very few detectable reads matching to the DcACV genome . This, along with the greater variance of DcACV incidence levels for all regions, shows the need for quantification of these viruses in future studies to compare how abundant they are in the ACP. DcFLV and DcPLV were also detected throughout most of the sampled citrus-producing counties, although not every month as was seen with DcACV, which suggests these viruses may exist at lower titers in the ACPs of these regions. Surprisingly, we also detected DcDNV throughout these vector populations, which has not been observed in Florida before this study . RemarkaRNAi defense mechanisms represent an integral part of the insect immune response to viral infections and allows for the substantial detection of viral sRNAs among total insect RNA extracted . The insHowever, it should be noted that our survey results show the detection of each ACP-associated virus when originating from a five-insect subset specimen (composite sample) of each monthly-collected county sample. This consistent number of insects for each population was intended to standardize possible incidence levels detected of each virus. If subsets of ACPs used for RNA extraction and cDNA synthesis were increased, incidences of these viruses may change and show different prevalence levels.Drosophila flies, and various other types of insects , in six Florida regional ACP populations, specific primers were designed based on the fragments obtained from bioinformatics analysis and used to screen additional D. citri populations, which were not analyzed by HTS  and multiple sRNA read detections using HTS established that DcACV was the most prevalent and widespread ACP-associated virus in Florida citrus groves. Following the completion of this study, our specific approach for targeting the mortality of the ACP as a biological control will undoubtedly influence the choice of viral vector we will venture forth with. On the one hand, our research findings provide robust evidence for DcACV as the likely choice to pursue as a possible viral candidate in RNA interference technology. Its clear ubiquity deserves future research into possible utilization as an RNAi delivery machinery against the HLB vector. On the other hand, the low prevalence and detection of DcRV may be considered as unfavorable to the ACP and could serve as an even better viral candidate for RNAi delivery. Regardless of the virus' abundance, any modified viral vector will require high durability in the insect to avoid losing efficacy, as it will be constantly subjected to evolutionary changes and potential suppression by the ACP. Detection of all the viruses in both nymph and adult ACP samples suggest the potential of vertical transfer of each virus from parent to progeny and each virus' capability to remain in the ACP populations. DcACV's consistent detection and prevalence in ACPs and its prevalence in detection through HTS also suggests the highest stability observed amongst the viruses during this study in the ACP virome. DcACV may not be treated as a foreign entity if manipulated and re-inoculated into wild Florida ACP populations, which strengthens its potential as an RNAi vector tool for future studies.CLas in these same ACP RNA samples used for the ACP-associated viral surveys. We also hope to determine the precise tissue localization of these ACP-associated viruses in the ACP, which will help strengthen future research by determining how efficient and effective this viral candidate's RNAi capabilities could be toward controlling the HLB disease cycle by killing the ACP.In conclusion, this comprehensive survey and the successful detection of two novel and five previously characterized ACP-associated viruses, coupled with further HTS analysis of these Florida ACP populations, have provided innovative and clearer insight regarding the spatial and temporal prevalence of these viruses in natural grove conditions. These results have greatly increased our knowledge of a potential source of viral candidates to use against this economically devastating insect vector in Florida. To capitalize on this study, our next steps will include establishing the presence and significance of /supplementary material.All datasets generated for this study are included in the articleKB performed the experiments, collected ACPs, analysed the data and drafted the manuscript. SG helped with RT-PCRs and PCRs, ACP collections, statistical analysis, and editing the manuscript. MR completed all the HTS. OB and AL received the funds, designed and directed the study. OB identified grove sites, collected ACP, purified virions, analysed the data and wrote the manuscript. All authors reviewed and edited the manuscript.Funding for this project was provided by the Citrus Initiative Grant of University of Florida IFAS and U.S. Department of Agriculture (USDA) under grant number 2015-70016-23011.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Post-maraviroc reversion of CXCR4-use to CCR5-tropic occurred in 7/8 participants with follow-up, suggesting selective maraviroc inhibition of CCR5-tropic variants in a mixed-tropic viral population, not emergence of de novo mutations in CCR5-tropic virus, as the main virologic escape mechanism. Maraviroc-resistant CCR5-tropic virus was observed in plasma from 5 treated participants with virus displaying reduced maximal percent inhibition (MPI) but no evidence of IC50 change. Env clones with reduced MPI showed 1–5 amino acid changes specific to each V3-loop region of env relative to Day 1. However, transferring on-treatment resistance-associated changes using site-directed mutagenesis did not always establish resistance in Day 1 virus, and key ‘signature’ mutation patterns associated with reduced susceptibility to maraviroc were not identified. Evolutionary divergence of the CXCR4-using viruses is confirmed, emphasizing natural selection not influenced directly by maraviroc; maraviroc simply unmasks pre-existing lineages by inhibiting the R5 virus. For R5-viral failure, resistance development through drug selection pressure was uncommon and manifested through reduced MPI and with virus strain–specific mutational patterns.Detailed clonal phenotypic/genotypic analyses explored viral-escape mechanisms during maraviroc-based therapy in highly treatment-experienced participants from the MOTIVATE trials. To allow real-time assessment of samples while maintaining a blind trial, the first 267 enrolled participants were selected for evaluation. At failure, plasma samples from 20/50 participants (16/20 maraviroc-treated) with CXCR4-using virus and all 38 (13 maraviroc-treated) with CCR5-tropic virus were evaluated. Of those maraviroc-treated participants with CXCR4-using virus at failure, genotypic and phenotypic clonal tropism determinations showed >90% correspondence in 14/16 at Day 1 and 14/16 at failure. Phylogenetic analysis of clonal sequences detected pre-treatment progenitor CXCR4-using virus, or on-treatment virus highly divergent from the Day 1 R5 virus, excluding possible co-receptor switch through maraviroc-mediated evolution. Re-analysis of pre-treatment samples using the enhanced-sensitivity Trofile HIV-1 entry into cells requires initial virus-cell contact via the CD4 receptor followed by co-receptor binding, most commonly through CCR5 (R5) . EmergenUnique among the current approved antiretroviral drugs, maraviroc (a CCR5 antagonist) binds with high affinity to host protein, CCR5, on the cell surface rather than to a viral protein . This cade novo mutation resulting in resistance in an R5 virus or through emergence of virus populations that use CXCR4 for entry (either through exposure of a pre-existing CXCR4-using minority virus population in the presence of maraviroc or through de novo mutation in an R5 variant enabling the use of CXCR4) [in vitro is a reduction in the maximal percent inhibition (MPI) rather than an increase in the 50% inhibitory concentration (IC50) [in vitro studies demonstrating that co-receptor switching is a significant evolutionary change requiring sequential accumulation of multiple mutations in the V1/2 and/or V3 regions of gp120 [There are two potential mechanisms of ‘viral escape’ from maraviroc: f CXCR4) –21. Consn (IC50) ,20. The n (IC50) ,23. Thisof gp120 .The safety and efficacy of maraviroc as a component of antiretroviral regimens for treatment-experienced persons infected with R5 HIV-1 was demonstrated in the MOTIVATE 1 and MOTIVATE 2 clinical studies ,26. In tWe examined the mechanisms of resistance to maraviroc in the MOTIVATE trials in detail to extend previous observations of CXCR4-using virus on-treatment and to investigate the origin of this virus (pre-existing or selected on-treatment) using clonal analysis to determine phenotype and phylogenetic relatedness. As participants were selected prior to unblinding the study, both maraviroc- and placebo-treated participants were included. In addition, R5 virus identified with reduced susceptibility to maraviroc was studied to establish whether resistance can be associated with any consistent mutational pattern in the V3-loop.The first 267 participants enrolled in the MOTIVATE 1 and 2 clinical studies were included in this study, and the analysis was performed after the last participant had reached Week 24 and while the study was still fully blinded . IndividThe exploratory analysis of mechanisms associated with virologic failure in the MOTIVATE 1 and 2 studies was carried out on a subset of the first 267 participants to be enrolled in the studies. Participants were categorized according to treatment status, viral load, tropism , and time of treatment failure. Participants excluded from the analysis comprised 138 who remained on treatment at Week 24 (Responders) with an HIV-1 RNA below the protocol-defined limit for running a tropism assay (<500 copies/mL) and 14 who discontinued treatment before Week 24 for reasons other than protocol-defined treatment failure. Filled (black) boxes indicate participants selected for analysis. DM/X4, CXCR4-using virus ; NR, no valid tropism result; R5, CCR5-tropic virus.3 . Further analysis of the sub-population indicated generally similar proportions of the 267 participants in treatment groups and with treatment failure as for the total population  and on-treatment (n = 48 clones) plasma HIV-1 RNA from each of the 20 selected participants with CXCR4-using viral populations underwent initial testing for co-receptor use with a simplified, single-well, pseudo-typed virus infectivity assay see . AdditioVirus from 5 of the 20 participants had a change in Trofile result between screening (R5) and Day 1 .For plasma from all 20 participants, a median of 104 functional Day 1 clones were observed per participant , providing a sensitivity to detect 2%-4% CXCR4-using clones with 95% probability. The V3-loop was successfully sequenced for the majority of Day 1 clones .Results from the infectivity analysis of clones were generally consistent with the Trofile findings. Among all 16 maraviroc-treated participants, the majority of clones showed R5-tropic phenotype at Day 1  but mostly R5-using phenotype , with overall 26% concordance. In addition, in clones from the Day 1 isolates from 2 of the 4 participants who received placebo, there was >97% concordance of genotype and phenotype tropism. Of the other 2 participants, T397 had only non-R5 clones detected using genotype but 61% R5 by phenotype, and in the Day 1 isolate from T57, the infectivity assay found R5 virus in 105 of 107 clones (98%), whereas genotype detected non-R5 virus in 105 of 107 clones (98%) .The infectivity analysis of clones on treatment was less comprehensive than at Day 1 because it was anticipated that the suppression of R5 virus by maraviroc would give rise to enhanced proportions of CXCR4-using virus. On treatment, a median of 23 functional clones  were analyzed successfully in the infectivity assay, sufficient to detect 9%-21% CXCR4-using clones with 95% probability. The V3-loop was successfully sequenced for the majority of clones .The on-treatment isolates from 15 of the 16 participants receiving maraviroc had a majority of non-R5 clones with both phenotypic and genotypic determinations . On-treatment clones from the other participant (T347) showed 65% R5 tropism using both phenotypic and genotypic determinations .Among the 4 placebo-treated participants, clones from the on-treatment virus had predominantly R5-tropic phenotype in 3 participants. Clones from the participant with the greatest proportion of R5 viral clones  included 2 of 20 (10%) X4 clones using both phenotypic and genotypic methods; this small proportion of virus with exclusively X4 tropism appears to have resulted in the phenotypic DM assignment at the viral population level, which is consistent with the previously demonstrated sensitivity to detect minor CXCR4-using variants .A confirmatory analysis using the original Trofile assay was performed on 11 to 12 selected clones from each of the time points for all 20 participants  who failed to respond to treatment, maintaining HIV-1 RNA 100,000 to 1,000,000 copies/mL and CD4 cell count <100 cells/mmA retrospective phenotypic re-analysis of tropism at Screening was performed using an enhanced sensitivity version of the Trofile assay (ESTA), which confirmed pre-existence of CXCR4-using virus at Screening in 13 of the 16 (81%) maraviroc-treated participants included in the clonal analysis, who were originally classified as having R5 virus using the Trofile assay . Thus, 8Eight of the 16 participants who received maraviroc and failed with CXCR4-using virus had at least one post-treatment virology follow-up sample. In 7 of these (87.5%), the virus reverted to R5 tropism after maraviroc was withdrawn, even if the exposure to maraviroc had continued for some time following first detection of CXCR4-using virus . Both individuals with detailed clonal analysis exemplified in Figs 50  with all viruses showing MPI ≥95% (Maraviroc susceptibility was assessed using the PhenoSense™ HIV Entry assay in virus from paired pre-treatment (Day 1) and failure (on blinded treatment) plasma taken from all 38 participants who failed treatment with R5 virus, regardless of whether they received maraviroc or placebo. Susceptibility results were obtained for all but 2 individuals . At Day 1, the R5 virus showed full susceptibility to maraviroc by ICMPI ≥95% .50 FC relative to the JRCSF reference strain of HIV-1 at the time of failure in the maraviroc or the placebo treatment groups . Viral load, CD4, and drug-treatment data for the other 3 participants receiving maraviroc and the participant who received placebo are provided in Reduced MPI were observed in viruses from 4 of the 11 maraviroc-treated participants and 1 placebo-treated participant who received open-label maraviroc following failure . The results were consistent with the population phenotypic findings, as MPI <95% was observed in all env clones from the treatment failure time points of all 4 maraviroc-treated participants, and the participant treated with open-label maraviroc after placebo, with reduced MPI in the population sample . Whilst 50 FC to maraviroc  did not show any reduction in MPI , and the susceptibility of the clonal virus was also in good agreement with the population data .Both Day 1 and on-treatment clones from the participant with a significant ICIn participants with reduced maraviroc MPI, those viral clones with reduced MPI were associated with 1 to 5 amino acid changes in the V3-loop relative to Day 1 . The majChanges in the V3 loop of viral clones from 4 participants during placebo treatment were also observed. Usually, there were only 0 to 2 amino acid changes, but in one individual (PID 18), a variant with 6 different amino acids was selected on treatment . This deIn order to determine whether the V3-loop amino acid changes observed following treatment with maraviroc were associated with reduced susceptibility and not unrelated changes as those seen in participants receiving placebo, site-directed mutagenesis (SDM) was performed. To assess whether the changes seen in the on-treatment samples of the 4 maraviroc-treated participants with reduced MPI were both sufficient and necessary to confer reduced susceptibility to maraviroc, SDM was used to insert the observed on-treatment amino acid variants into a Day 1 derived viral clone or to remove it from the on-treatment variant clones to re-establish the Day 1 V3-loop sequence .In cloned virus from 2 of the 4 participants (PID 3 and PID 8), the changes observed in the V3-loop appeared to be necessary and sufficient to establish reduced MPI –based assay .Individual env clones with V3-loop genotypes similar to the consensus sequences were selected from maraviroc-treated participants with reduced MPI or ICed assay . There wed assay . At failCross-resistance with another CCR5 antagonist, aplaviroc (APL), was assessed in 4 of the 5 participants with reduced MPI. Only virus from PID 3 showed possible cross resistance with aplaviroc . The aplThe MOTIVATE 1 and 2 studies provided a unique opportunity to examine the viral changes associated with failure in highly treatment-experienced participants receiving a maraviroc-containing regimen. Many received sub-optimal background regimens as they had limited remaining treatment options due to previous exposure to multiple regimens and development of resistance ,26. In mFor the MOTIVATE studies and other early maraviroc studies, the original Trofile assay  was usedIn the phase 2a maraviroc monotherapy studies, A4001007 and A4001015, CXCR4-using virus was detected in 2 of 62 participants after 10 days of treatment . Phylogein vitro and were often considered to be non-functional in the Trofile assay. Indeed, CXCR4-using virus is generally understood to be at a disadvantage relative to R5 virus in infected persons; CXCR4-using virus is rarely transmitted , but isiewed in . Under niewed in ,42 and diewed in ,44.The evolution of CXCR4-using virus has been associated previously with increased disease progression when it occurred in untreated or sub-optimally treated individuals during the natural course of infection ,46. Howe50  has been observed rarely. Reduced MPI to maraviroc was the key indicator of viral resistance, which is consistent with the virus developing the ability to enter cells through maraviroc-bound CCR5 [Consistent with maraviroc being an allosteric inhibitor of CCR5 binding, among participants who failed with R5 virus, a change in ICund CCR5 . MPI redund CCR5  and for und CCR5 . In all,The mechanism for selection of ‘true’ resistance to maraviroc in R5 virus is for the virus to adapt to be able to bind to the CCR5 molecule with inhibitor present, with greater binding through the CCR5 N-terminal region in the absence of maraviroc . HoweverWhile the overall molecular mechanism to attain HIV envelope binding to CCR5 appears to be the same for different maraviroc-associated resistance , the exact genotypic changes required seem to be highly variant. Although changes in the V3-loop were observed on all resistant viruses in the present study, there was no consistent pattern of mutations; indeed, regions outside the V3-loop might be contributory in addition to compensatory . StrikinA separate analysis has been performed using frozen plasma samples from 8 MOTIVATE participants who experienced treatment failure with R5 virus that had reduced MPI to maraviroc . Four ofThe X-ray crystal structure of maraviroc-bound CCR5 has recently been solved, confirming that the compound binds deep into the bottom of the pocket formed by the helices of CCR5, and is consistent with it being an allosteric inhibitor of viral entry . FurtherIn conclusion, extensive data from phenotypic and genotypic analysis of viral escape from inhibition by maraviroc is presented at a clonal level. The main virologic change observed in virologic failure samples was the detection of CXCR4-using virus. This was due in large part to unmasking of pre-existing CXCR4-using virus populations caused by R5 virus inhibition with maraviroc. The effect was reversible on stopping maraviroc treatment. Reduction of maraviroc susceptibility in R5-tropic virus was less frequent and associated with various changes in the env protein V3-loop, but without a common resistance pathway.® assay  [MOTIVATE 1 and 2 were randomized, double-blind, placebo-controlled, phase 3 trials conducted in Canada, the United States, Australia, and Europe to assess the safety and efficacy of maraviroc in combination with other antiretroviral drugs for the treatment of HIV-1 . The studies have been described in detail elsewhere, but briefly, eligible participants had an R5 HIV-1 screening tropism result using the original TrofileCA, USA) ,26. The Treatment failure was defined as 1 of 4 virologic endpoints confirmed by a second consecutive measurement within 14 days:increase in HIV-1 RNA to ≥3 times the Day 1 level at or after Week 2;10 copies/mL at or after Week 8;decrease of <0.5 log10 copies/mL at or after Week 8, after a decrease of ≥2.0 log10 copies/mL; anddecrease of <1.0 logincrease in HIV-1 RNA to ≥5000 copies/mL after a decrease to <400 copies/mL had been recorded on 2 consecutive visits.Treatment failure was categorized as ‘early’ failure, occurring at or before Week 8, or ‘late’ failure, occurring after Week 8. Participants who did not experience treatment failure were categorized as responders.An exploratory analysis of tropism changes and maraviroc resistance was carried out on a subset of participants selected on a blinded basis using a pre-specified ~6-month period of enrollment that would give ~250 participants for analysis through Week 24. A data cut after all completers had reached Week 24 was performed. This allowed for a detailed observational analysis of the mechanisms of virologic escape from maraviroc including phenotypic and genotypic clonal analysis in order to provide a timeous assessment of mechanisms of virologic escape to inform clinical use of maraviroc.All participants selected for analysis were tested for tropism at treatment failure or Week 24. Those with CXCR4-using virus were subjected to random selection of 20 participants on a fully blinded basis. Participants were categorized by time to failure . As there were only 6 participants with non-B viral infection failing with CXCR4-using virus, these were omitted from the analysis in case their findings might bias the analysis. A third party, which could be unblinded, was provided with participant identifier numbers in groups according to time to failure  and Day 1 tropism. A random selection from each of the 6 groups was then made according to a set of pre-defined rules to ensure proportionate representation according to the number of participants in each group.Co-receptor tropism in the MOTIVATE studies was determined using the original Trofile assay, as previously described . BrieflyA retrospective analysis of the samples was performed using the enhanced sensitivity Trofile assay (ESTA). This assay follows the same procedures as the original Trofile assay described above but has undergone further optimization to increase sensitivity to detect minor X4-tropic variants in the population when present with 0.3% prevalence .Clones from paired pre-treatment  and on-treatment samples from selected participants were isolated and pre-screened for co-receptor tropism using a high through-put infectivity assay. There are several differences between the Trofile assay used for clinical testing and the infectivity assay used to screen clones in this study. A key difference is the lack of confirmation of viral entry specificity using a co-receptor inhibitor. In addition, single-well, low-volume measurements and potential cross-talk between wells limit the ability of the infectivity to mirror the performance of the Trofile assay. Published information and a simple statistical model were used to decide how many clones to pre-screen for tropism . Based ohttp://coreceptor.geno2pheno.org/index.php, accessed February-April 2013). The consensus sequences were calculated using >80% identity. Less than 80% was represented by ‘X.’A region including the V3-loop was sequenced for all clones  and the sequences aligned using the CLUSTAL_X program . The V3-A further confirmatory re-analysis of tropism was performed using the clinical version of the original Trofile assay. This analysis included 12 clones each per paired Day 1 and on-treatment samples. Samples were selected such that any clones with discordance between genotypic and phenotypic findings from the infectivity assay were included where possible.DNA sequences of minimum length 280 nucleotides from each env clone were used to infer evolutionary trees. Reference sequences from unrelated individuals were included to root the trees. For each participant, 7 reference sequences were also included in the alignment:The reference HIV-1 strain, HXB2 (NCBI accession number K03455), was used as an out-group  to root the phylogenetic trees.In addition, an R5 env clone and CXCR4-using env clone from 3 other participants were used to represent examples of clade B viruses currently in circulation.http://www.megasoftware.net/home) to collapse large branches and enable visualization and annotation; bootstrap values ≥75 are shown.The nucleotide sequences of interest and the seven reference sequences were aligned using the CLUSTAL_X program  and defaVirus susceptibility to maraviroc was determined using two different methods; the PhenoSense™ HIV Entry assay  and a PBL based assay, as previously described ,19,21,3050 relative to the reference virus JRCSF ; or a >2-FC in IC50 between virus taken at Day 1 and virus taken at treatment failure from the same participant.The PhenoSense HIV Entry assay is a modification of the Trofile assay. Maraviroc susceptibility is determined from dose-response plots of percent inhibition of viral replication versus drug concentration using the CCR5-expressing U87CD4+ cells. Pre-clinical studies have shown maraviroc resistance can be identified by plateaus in maximum percent inhibition (MPI) in the PhenoSense HIV entry assay . Plateau50 FC relative to Ba-L and arithmetic mean percentages in MPI. A shift in IC50 FC was only deemed relevant if the dose-response curve showed no evidence of a plateau in MPI. The accuracy of the estimates of susceptibility to maraviroc (IC50 FC and MPI) in PBL assays was reported by calculating 95% confidence intervals. Thus, it was possible to distinguish between dose-response curves that reached close to 100% inhibition  from those curves that demonstrated a plateau with MPI<100%. In this way, MPI values <95% were considered to reflect a biologically significant reduction.For the PBL-based assay, participant-derived env genes from the vectors used in the PhenoSense HIV Entry assay were transferred into an NL4-3 vector that was transfected into HEK-293 cells to produce replication-competent virus able to infect PBL. Phytohemagglutinin-stimulated PBL (pooled from multiple donors) were acutely infected with the replication-competent virus and cultured in the presence or absence of maraviroc for 7 days before estimating the amount of replication in each well by measuring reverse transcriptase (RT) levels in the culture supernatant. The CCR5-tropic lab strain Ba-L was included in all PBL assays as a reference strain; maraviroc susceptibility was reported both as geometric mean ICIn order to identify key genotypic markers of maraviroc resistance, the V3-loop amino acid residues of 4 treatment failure maraviroc-resistant clones were back mutated to correspond to the genotype of their respective Day 1 env clones. Furthermore, the Day 1 clones were also mutated to the sequence of their respective maraviroc-resistant counterparts.Plasmid DNA corresponding to the Day 1 and treatment failure env clones from the participants was provided by Monogram Biosciences, Inc. Each env gene was transferred to an appropriate shuttle vector (pSP72 or pSP73) prior to SDM using the Quick Change II XL Site Directed Mutagenesis Kit  and sequence specific oligonucleotide primers. DNA was isolated from bacterial cultures using QIAprep Spin Miniprep Kit (Qiagen) and sequencing was performed by Lark Technologies, Inc. Sequence analysis using Vector NTI software  confirmed the authenticity of the SDM sequences. In each case, the mutated sites were those that were consistently associated with the maraviroc-sensitive or maraviroc-resistant clones sequenced for each pair.The mutated env clones were then tested for susceptibility to maraviroc, aplaviroc, and enfuvirtide by Monogram Biosciences, Inc, in the PhenoSense HIV-1 entry assay as described above.50 fold-change at failure and between treatment groups. The P value and mean difference with 95% confidence intervals are quoted.Analysis of variance (ANOVA) was used to identify changes in maraviroc ICThe following HIV-1 envelope sequences have been submitted to GenBank:Maraviroc-treated: KT452383-KT452406, KT452431 –KT452454, KT452263-KT452310, KT452479 –KT452502, KT452527 –KT452562, MG183941 –MG188267, MG244243-MG244246.Placebo: KT452599-KT452622, KT452359-KT452382, KT452084-KT452107.S1 Checklist(DOC)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 FigEach tree is rooted using HXB2 (NCBI accession number K03455). Examples of 2 clonal sequences: R5 (Day 1) and CXCR4-using (on-treatment) from 3 unrelated individuals were included together with the patients’ Day 1 and on-treatment nucleotide sequences. CLUSTAL_X was used to infer the neighbor-joining tree and the diagrammatic representation was created using MEGA, collapsing the branches with the size of the triangle representative of the number of sequences in the branch and the bootstrap values from ‘100 trials’ added to the nodes. Only those values greater than 75 were included. Green triangles and lines represents sequences from R5 clones and blue triangles and lines those from CXCR4-using clones. If the branch contained a mix of R5 and confirmed CXCR4-using clones, then that branch and triangle are colored blue. Black lines represent non-functional clones where the tropism could not be determined phenotypically. Individual clones not clustering in the main lineages are represented by single lines. Sequence diversity is represented by horizontal distance.(PDF)Click here for additional data file.S3 FigEach tree is rooted using HXB2 (NCBI accession number: K03455) and inferred using PAUP with the HKY + gamma model of nucleotide substitution and NNI heuristic settings. Day 1 clones are labeled D and in black font. On-treatment clones are labeled E and in blue font.(PDF)Click here for additional data file.S4 Fig(A) Change in viral load (◊ dashed line) and CD4 (▪ solid line) for participant 8, who received maraviroc in combination with didanosine (ddI), tenofovir (TFV), lopinavir/r (LPV/r), and enfuvirtide (ENF) for 27 weeks (shaded area) and failed therapy at Week 24  with R5 virus. Regimen prior to Day 1: ddI, LPV/r, ENF. OBT from Day 1: ddI, TFV, LPV/r, ENF. Screening: ddIsens, TFVsens, LPV/rres, ENFres (V38A). Failure: ddIres, TFVres, LPV/rres, ENFres (V38A). (B) Change in viral load (◊ dashed line) and CD4 (▪ solid line) for participant 11, who received maraviroc in combination with emtricitabine (FTC), tenofovir (TFV), stavudine (d4T), and enfuvirtide (ENF) for 15 weeks (shaded area) and failed therapy at Week 8  with R5 virus. Regimen prior to Day 1: FTC, TFV, d4T, TPV/r, ENF. OBT from Day 1: FTC, TFV, d4T, RTV, ENF. Screening: FTCres, TFVsens, d4Tsens, RTVres, ENFres (N43D/N). Failure: FTCres, TFVsens, d4Tres, RTVres, ENFres (N43D/N). (C) Change in viral load (◊ dashed line) and CD4 (▪ solid line) for participant 12, who received maraviroc in combination with didanosine (ddI), tenofovir (TFV), amprenavir (AMP), and enfuvirtide (ENF) for 12 weeks (shaded area) and failed therapy at Week 8  with R5 virus. Open-label therapy with maraviroc commenced Week 21 and finished Week 60 (shaded area). Regimen prior to Day 1: ddI, TFV, AMP/r, ENF. OBT from Day 1: ddI, TFV, FPV/r, ENF. Screening: ddIres, TFVres, FPV/rres, ENFres (N43D). Failure: ddIres, TFVres, FPV/rres, ENFres (N34D). (D) Change in viral load (◊ dashed line) and CD4 (▪ solid line) for participant 16, who received placebo in combination with abacavir (ABC) and tenofovir (TFV) for 14 weeks and failed therapy at Week 8  with R5 virus. Open-label therapy with maraviroc commenced Week 24 and finished Week 60 (shaded area). Regimen prior to Day 1: 3TC, TFV, ATZ/r. OBT from Day 1: ABC, 3TC, TFV. Screening: ABCsens,* 3TCres, TFVsens.* Failure: ABCsens,* 3TCres, TFVsens.*. *Sensitive by phenotype but resistant by genotype.(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S1 Protocol(PDF)Click here for additional data file.


Although parasites are still endemic in developing areas, residents in those regions seem not to be affected by the presence of intestinal protozoans. This study aimed to investigate whether pathogenic and commensal protozoans are the causal agents of diarrhea via a school-based cross-sectional survey conducted in Indonesia, in September 2016.Giardia intestinalis (56.3%), Entamoeba histolytica (0%), E. dispar (6.9%), E. moshkovskii (0%), E. hartmanni (31.3%), and E. coli (44.4%). Observational evaluation of stool conditions using the Bristol stool chart confirmed the loose stool rate (33.3–90.9%) in each age group. Logistic regression analysis of protozoan infection or colonization for loose stool (mild to severe diarrhea) as an outcome revealed no significant findings in examined protozoans including pathogenic G. intestinalis infection , except in E. hartmanni colonization .Molecular screening for intestinal protozoans in collected 144 stool samples from healthy students (age range 7–15 years) was carried out. The prevalence of protozoan parasites was as follows: G. intestinalis could regard commensal, while E. hartmanni seems to possess a certain pathogenicity as a causal agent of mild diarrhea.The molecular survey of intestinal protozoans targeting healthy population with their stool form evaluation could address the pathogenicity of those parasites appropriately. In comparatively higher-age children at least 7 years of age or greater in the endemic area,  Giardia intestinalis  infection is still highly endemic in most developing regions   and ass. B (54.2%)], E. histolytica (0%), E. dispar (6.9%), E. moshkovskii (0%), E. hartmanni (31.3%), and E. coli (44.4%)  [ass. A (69.6%) and ass. B (65.4%)], E. dispar (80.0%), E. hartmanni (68.9%), and E. coli (64.1%)  with those genotypes [ass. A  and ass. B ], except in E. hartmanni colonizations .Results of logistic regression analysis of protozoan infections or colonizations for diarrhea (loose stool form) as outcome Table  revealedThe stool form evaluation targeting healthy population could address the loose stool condition, which means mild to severe diarrheal conditions in those individuals Fig. . ObserveG. intestinalis infections were described [G. intestinalis infection and loose stool, which indicates mild diarrheal status, was evaluated. The loose stool rates among G. intestinalis infected individuals were almost of the same level as those in commensal non-pathogenic Entamoeba spp.-colonized ones, and the correlation between the mild diarrheal stool condition and G. intestinalis infection could not be confirmed  and a mean duration of cysts excretion 7.18 weeks), for escribed . On the 8 weeks, E. hartmanni colonization was observed  for the analysis. This means that even E. hartmanni possess a certain pathogenicity, though the level of pathogenicity might be limited to mild. Meanwhile, the presence of recently proposed potential pathogens such as E. moshkovskii and D. fragilis together with the possible pathogenicity of E. hartmanni emphasize the need of further study on pathogenicity evaluation for commensal protozoan parasites that have been neglected so far.Possible pathogenicity related to the ed Table ; howeverdiarrhea  and reacdiarrhea . AlthougDue to the lack of evaluation of viral and bacterial pathogens for diarrhea, the results of this study might be considered to be preliminary, and further in-depth research is clearly required to understand the findings.G. intestinalis and Entamoeba species. We found that only E. hartmanni colonization was associated with loose stool, though no correlation was found with G. intestinalis infection. In children living under a parasites-endemic area in Indonesia, E. hartmanni could be a mild pathogen, whereas G. intestinalis might be regarded as commensal, though the burden on children under 7 years of age may not be negligible.We molecularly examined occult protozoan infections in healthy school children in Indonesia to evaluate the real pathogenicity of


Arabidopsis making it the largest family of plant receptor kinases (RKs). Genetic and biochemical studies have provided valuable data regarding peptide ligands and LRR-RKs, however, visualization of ligand/LRR-RK complex structures at the atomic level is vital to understand the functions of LRR-RKs and their mediated biological processes. The structures of many plant LRR-RK receptors in complex with corresponding ligands have been solved by X-ray crystallography, revealing new mechanisms of ligand-induced receptor kinase activation. In this review, we briefly elaborate the peptide ligands, and aim to detail the structures and mechanisms of LRR-RK activation as induced by secreted peptide ligands within plants.In recent years, secreted peptides have been recognized as essential mediators of intercellular communication which governs plant growth, development, environmental interactions, and other mediated biological responses, such as stem cell homeostasis, cell proliferation, wound healing, hormone sensation, immune defense, and symbiosis, among others. Many of the known secreted peptide ligand receptors belong to the leucine-rich repeat receptor kinase (LRR-RK) family of membrane integral receptors, which contain more than 200 members within  Arabidopsis genome [Plant development is mostly a post-embryonic event in which intercellular signaling and cell-cell communication play a large role . In reces genome . Many ofs genome . Ligand s genome ,5.Arabidopsis is considered to be the largest family of plant receptor kinases [Based on their ectodomain structures, plant RKs can be classified into 14 subfamilies ,6. The l kinases . The LRR kinases . LRR-RKs kinases . Both LR kinases .Arabidopsis thaliana genome is predicted to encode 11 LRXs [Arabidopsis are expected to be involved in regulating proper root hair cell morphogenesis and elongation [Apart from LRR-RKs and LRR-RLPs, another class of LRR containing proteins is present in plant cells known as LRR-extensins (LRXs) which are localized within the cell wall. The  11 LRXs . StructuLRR-RKs have been studied extensively with a large quantity of data concerning peptide ligands and LRR-RKs having been obtained from genetic and biochemical experiments ,13,14,15Arabidopsis thaliana, studies predict that more than 1000 genes may encode secreted peptides [Eukaryotes use a variety of peptides and steroids to mediate intercellular communication. In mammals, peptides act as hormones and growth factors that take part in multiple signaling pathways to regulate various cellular processes . In planpeptides . These fMost of the plant LRR-RLK ligands are part of either paracrine or autocrine signaling mechanisms. In the case of the paracrine signaling system, the diffusible molecule released from one cell can act on neighboring target cells. The best example of paracrine signaling system is the CLV3 ligand, which is expressed in the central zone of shoot apical meristem (SAM), and can exert its effects within a short range of cells . In the Within plants, secreted peptides act extracellularly and are recognized by the transmembrane receptors present on the neighboring cells and can regulate cellular signaling pathways. Previously plant secreted peptides were classified into two major groups: small post-translationally modified peptides and cysteine-rich peptides (CRPs). Mature post-translationally modified peptides are smaller, ranging from 5 to 20 amino acids . On the A majority of the plant peptides are derived from a non-functional pre-protein that both gets proteolytically processed and/or enzymatically modified to produce biologically active peptides . HoweverIn this review, we will focus mainly on the peptide ligands and their corresponding LRR-RLKs that have been structurally characterized.Proteolytic processing is an integral step in producing small post-translationally modified peptides. This process involves a large precursor polypeptide containing an N-terminal signaling sequence which is translated and subsequently cleaved by proteases and modified to yield a smaller mature functional peptide. Major post-translational modifications that occur on these peptides include sulfation of tyrosine, hydroxylation of proline, and arabinosylation in plants . HydroxyThe hydroxyproline residues of peptides like CLV3, CLE9, and CLE2 are further modified by the addition of L-Arabinose sugar moiety ,86,90. HArabidopsis, the CLE family of small secretory proteins can be processed to 12–13 amino acid residue peptides with two conserved proline residues at positions four and seven that are usually hydroxylated [CLE genes, including CLV3, which express proteins smaller than 15 kD [CLAVATA3 (CLV3)/endosperm surrounding region (CLE) family of peptides is one of the most studied families of small post-translationally modified peptides. In oxylated . In the oxylated . Whetheran 15 kD ,92. AlonCLE genes, were applied to Arabidopsis seedlings, 18 of them were shown to arrest root growth. These 18 synthetic peptides include CLV3 and are classified as A-type. The remaining four peptides, CLE41p, CLE42p, CLE43p, and CLE44p do not influence root growth and are classified as B-type [In contrast, the CLE domain located at the C-terminus is highly conserved. Intriguingly, the middle non-conserved region is mostly dispensable for their in vivo function . It was s B-type .Studies of these two groups have shown that A-type CLE peptides function to define the balance between stem cell proliferation and differentiation in Shoot Apical Meristem (SAM) or Root Apical Meristem (RAM) ,93,94, wArabidopsis, soybean, and common bean contain 32, 84, and 44 CLE peptide-encoding genes respectively [Arabidopsis show that the pre-pro-peptide CLEs could be grouped based on their functions and CLE domains. The CLE pre-pro-peptides were classified into seven distinct groups (Groups I–VII) [Arabidopsis [ectively . Bioinfos I–VII) . This nebidopsis . In thisbidopsis .The LRR-RK protein CLVAVATA1 (CLV1) has been identified as the plasma membrane receptor for CLV3 ,14,71,99CLE genes CLE41 and CLE44 both encode the same B-type CLE peptide TDIF, which is hydroxylated and proteolytically processed to a 12 amino acid residue peptide TDIF [Two ide TDIF   also originates from a proteolytically cleaved non-functional precursor . IDA pepCRPs can be characterized by the presence of a conserved N-terminal region that includes a secretion signal sequence and a C-terminal cysteine-rich region possessing 4–16 cysteine residues. Some CRPs like RALFs and STOMAGEN require to be proteolytically processed to produce the mature, biologically active peptides ,134 Fig. CRPs arDuring stomatal development, the “one-cell-spacing rule” is enforced by short distance communications between neighboring cells, which is governed by the specific interactions between the secreted EPFL family cysteine-rich proteins, as well as the ERECTA family of LRR domain-containing transmembrane receptors (ERfs) ,143. BotMedicago or Arabiodpsis [Arabidopsis, these peptides can act either in a tissue-specific manner, induced by pathogens or wound response. In peas, two genes encode two CRPs, ENOD3 and ENOD14 [Arabidopsis [DEFENSIN peptides also belong to the CRPs and have been found to resist a variety of plant pathogens. These antimicrobial peptides are small (5–6 kDa) in size with more than 300 DEFENSIN peptides being identified within biodpsis ,162. In d ENOD14 . These Cbidopsis ,165,166.bidopsis . It is aTorenia fourneri and were shown to be involved in micropylar pollen tube guidance. Mature LUREs from Torenia species are 62–70 amino acids long and contain six cysteine residues [Arabidopsis thaliana, five LURE peptides (AtLURE1.1–AtLURE1.5) have been identified as attractants to regulate pollen tube guidance. It has been shown that all AtLURE peptides are capable of attracting pollen tube except for AtLURE1.5 which has only five cysteine residues instead of six [Arabidopsis thaliana as the cell surface receptors to perceive AtLURE1.1 and AtLURE1.2 signaling molecules [Arabidopsis have been implicated in the LURE-mediated signaling pathway [In tomatoes, some CRPs such as LAT52, LeSTIG1 have been identified to play important roles in pollen tube germination, pollen tube elongation, etc. LAT52 interacts with two specific pollen receptor kinases LePRK1 and LePRK2 ,168. Anod of six . Pollen olecules . Additio pathway .Arabidopsis thaliana, Arabidopsis helleri, and Arabidopsis lyrata. These peptides are capable of attracting prk6 mutated pollen tubes, implying that their signaling cascade does not require PRK6 as a receptor [Recently, XIUQIU cysteine rich peptides have been identified, which can attract pollen tubes in a non-species-specific way . XIUQIU receptor . HoweverArabidopsis and other LRR-RKs [Bacteria, yeast, animals, and humans all contain proteins with LRR motifs . Animals LRR-RKs .Plant LRR-RK ectodomains are made up of 1–30 individual leucine-rich repeats; each repeat is generally 20–29 amino acids long . The plaThe N- and C-terminal regions of LRR ectodomains contain hydrophilic capped regions that mask the hydrophobic ends from solvent accessibility . The capMany LRR-RK extracellular domains have been structurally characterized. These structures have provided useful information about receptor-ligand interactions, receptor-co-receptor complex formations, and the structural rearrangements that lead to kinase domain activation ,182,183.Arabidopsis thaliana belong to the small LRR ectodomain family  and Pollen Receptor Kinase 6 (PRK6) LRR ectodomain structures have been determined with PRK3 resembling closely the SERK family members  and subsequently being grouped into the small LRR ectodomain family [The large LRR extracellular domains usually have 20–29 LRR repeats that form a twisted superhelical structure to bind ligands. The small LRR proteins, in contrast to large LRR ectodomains, contain mostly five LRRs that fold as a small arc-shaped structure . Recentln family   and Golgi, in which a multi-subunit enzyme and an oligosaccharyl tranferase complex governs the transfer of a core N-glycans (Glc3Man9GlcNac2) to the nascent polypeptide chains [In plants, many secreted proteins, cell surface receptors, as well as membrane and cell wall-associated proteins are frequently modified with complex glycan structures. Two major types of glycosylation can occur: N-linked glycosylation on the amide group of the asparagine residues and O-linked glycosylation on the hydroxyl group of either the serine, threonine, or hydroxylproline residues ,197,198 e chains ,200.N-glycosylation happens on specific asparagine residues within the consensus sequence of Asn-X-Ser/Thr (NXS/T motifs) where X can be any amino acid except proline and aspartate [N-glycans reside on the exposed surface of the protein and form flexible extended branches [N-glycosylation ensures not only proper folding of the conjugated proteins but it also plays a significant role in various biological processes such as maintaining protein conformation and stability, executing cellular interactions, and protecting from protease degradation [Arabidopsis thaliana that most of the N linked glycans are of complex type [N-glycosylation in the ER and Golgi bodies can severely affect the abiotic stress tolerance in Arabidopsis [spartate ,194,196.branches . N-glycoradation ,203. Mamradation ,205. So lex type . Impropebidopsis ,208.N-glycosylation motifs such as N(X)S/T, have been found on the extracellular domains of the LRR-RKs in Arabidopsis thaliana. FLS2, EFR, TDR, PSKR1, BAK1, PRK3, PRK6, ERL1, and MIK1 all contain putative N-glycosylation motifs. The expressed recombinant ectodomains of LRR proteins are often larger than their predicted molecular weights, as shown in the recombinant ERECTA ectodomain protein which has a molecular weight (MW) of approximately 70 kD and is about 10 kD larger than its predicted MW of 60 kD. Similar discrepancies in molecular weights have been found in TDR, PRK3, MIK1, etc., indicating that these proteins were likely glycosylated when expressed in a heterologous expression system. The crystal structures of HAESA, ERL1, TDR, PSKR1, and PRK3 ectodomains also contain some N-linked glycans [Several conserved  glycans ,19  to be heavily glycosylated . Characterization of these glycosylation moieties will shed light onto their proper function. The Glyco-EP bioinformatics program has predicted the N-linked and O-linked glycosylation sites on the ectodomains of the ERfs and TMM  Table 2Table 2.N-glycosylation motifs N(X)S/T are predominantly present within the α-helical region located on the convex side of the LRR domain and are essential for the proper folding of the overall structure [N-glycosylation in plant LRR-RK function. In Arabidopsis, EFR recognizes bacterial EF-Tu by interacting with a Pathogen Associated Molecular Pattern (PAMP) molecule elf18. It has now been found that N-glycosylation is essential for this recognition [It has been reported that the putative tructure . Severalognition .N-glycosylation motifs were altered, it was observed that plants became more susceptible to bacterial infection and developed disease symptoms [N-glycosylation sites may impede the EFR-elf18 interaction and downstream signaling networks [N-glycosylation sites [When some of the conserved symptoms . Based onetworks . N-143 anetworks . It has on sites .N-glycosylation in LRR domain folding and stability. Furthermore, it is evident that N-glycans significantly contribute to LRR-Ligand binding activity, function, and the elicited downstream signaling network.Interestingly, some reports show that under-glycosylated EFR, and non-glycosylated FLS2, both fail to form efficient ligand binding sites . MutatinFLS2 is an LRR-RK that senses a 22 amino acid long conserved epitope of bacterial flagellin flg22. It is the first reported structure that provides critical biophysical and biochemical information of a peptide ligand interacting with a plant LRR-RK ,16,212. Arabidopsis thaliana Pep1 is a 23 amino acid long peptide produced in response to pathogenic attacks and is perceived by the LRR-RKs PEPR1 and 2 [R1 and 2 ,214,215.R1 and 2 . AtPep1 R1 and 2   play essential roles in mediating intercellular communications in most multicellular organisms. The binding of ligands to their extracellular domain(s) usually induces activation of RKs, which triggers either oligomerization or conformational changes of the RKs, their intracellular kinase domains are then activated to initiate signal transduction into the cells ,222,223.Arabidopsis genome [Gene duplication and functional divergence has created the SERK family of small LRR-RKs ,56. So fs genome . The SERs genome ,187,188 s genome . Studiess genome ,187,188.Kinase domains of large LRR receptors can physically interact with BAK1, and the SERK family of co-receptors, but their exact structural configuration remains elusive. The kinase domain interaction, therefore, proves the notion of ligand-induced heterodimerization mediated RK activation in plants. SERK family members also act as co-receptors for PSKR1 and TDR, etc. A similar kind of co-receptor binding site is observed in the PSKR1-SERK1 ectodomain complex. The PSK ligand promotes PSKR1-SERK1 interaction, thereby supporting the ligand-mediated heterodimerization model. Surprisingly, it has been found that PSK does not act as the “Molecular Glue” in this scenario; instead, the principal function of PSK is to stabilize the PSKR ID region, which in turn interacts with SERK1 by forming a stable heterodimeric complex ,186. ThiThe flg22-FLS2-BAK1 structure involves the only heterodimerization with no higher order oligomerization event observed . This agBased on multiple studies, it has been hypothesized that ligand-induced heterodimerization with a co-receptor is a crucial mechanism for the intracellular kinase activation in plants. Studies on plant LRR-RKs have shown that various ligands can strongly promote the interaction between the ectodomain of a receptor and its co-receptor. These strong interactions result in the formation of a heterodimeric complex that brings the transmembrane and kinase domains of the corresponding receptor-co-receptor together. The kinase domains of these RKs go on to interact and form a dimeric conformation for their activation ,228.LRR containing proteins have been identified in archaea, bacteria, eukaryotes and even in viruses . LRR proThe evolutionary history of LRR proteins is still unknown. Kobe and Deisenhofer predicted that LRR proteins might have a bacterial origin while also hypothesizing that LRR proteins may have evolved before the eukaryotes even appeared ,229. HowIt is well known that plant LRR-RKs regulate a variety of cellular processes inside plant cells. LRR-RKs control multiple signal transduction pathways leading to cell division, cell differentiation, stem cell balance, immune and pathogen response, symbiosis, plant development, pollen tube guidance, and ovule targeting ,167,169.Although the overall LRR-RK structures are relatively similar, their corresponding ligands are very much diverse. Structures of LRR-RKs in complex with small peptides, CRPs, and other small molecules have been described in much detail with several structural models having clearly shown that ligand binding induces the kinase domain activation to start the subsequent downstream signaling cascade. It has been frequently observed that ligand binding induces a heterodimerization event with a small co-receptor kinase or an LRR-RLP which is devoid of a kinase domain. However, our understanding of plant RK mediated signaling is still limited with many questions remaining to be addressed.At first, it is unclear whether the SERK family members only function as the universal co-receptors or whether they have any other intrinsic functions. It is possible that some other proteins can also act as co-receptors and mediate receptor kinase activation. Previously, we have reported that AtPRK3 is structurally similar to the SERK family members. Based on structural similarity, sequence conservation, and a conserved surface patch on the surface of AtPRK3, we hypothesized it to serve as a co-receptor . It is pMoreover, AtPRK3 and AtPRK6 share more than 60% sequence identity, and only AtPRK6 can recognize the AtLURE1.2 peptide. It will be interesting to address the question in relation to AtPRK3 being unable to interact with AtLURE1.2. Is there any other unidentified ligand present that interacts with AtPRK3 or can AtPRK3 only act as a co-receptor? Secondly, as for the functionality of LRR-RLPs in receptor kinase signaling, so far, it has only been demonstrated that TMM acts as a specificity switch to recognize the correct ligands . It is sCellular and genetic studies on TDR, CLV1, and ERECTA have revealed that the downstream signaling components are diverse. However, there is very scarce information about the downstream signaling components available so far ,239,240.Arabidopsis thaliana, LRR-RKs have been classified into 12 subgroups. Five subgroups are present in Oryza sativa, and 14 subgroups in Populus trichocarpa [In recent years, multiple LRR-RK ectodomain structures have been determined, but studies on the structures of their KDs are lacking. It is widely considered that the KDs are relatively conserved and contain 12 subdomains that fold into a two-lobed structure ,242. Phychocarpa ,244. FroSubstantial available genetic, phylogenetic, cellular, and structural information about the LRR-RKs has improved our knowledge about LRR-RK evolution, ligand perception, and plant signaling. Further studies are still required to understand the evolution, cellular, molecular, and mechanistic basis of the LRR-RKs mediated plant signaling pathways as a whole. A unified theory based on this information will pave our way to fully understanding plant growth, development, and their interactions with the environment.


Aspergillus flavus is known to be infected with mycoviruses. In this study, we report a novel mycovirus A. flavus partitivirus 1 (AfPV1) that was originally isolated from the abnormal colonial morphology isolate LD-3-8 of A. flavus. AfPV1 has spherical virus-like particles about 40 nm in diameter, and three double-stranded RNA (dsRNA) segments  were packaged in the virions. dsRNA1, dsRNA2, and dsRNA3 each contained a single open reading frame and potentially encoded 62, 42, and 32 kDa proteins, respectively. The dsRNA1 encoded protein shows similarity to the RNA-dependent RNA polymerase (RdRp) of partitiviruses, and the dsRNA2 product has no significant similarity to any other capsid protein (CP) in the GenBank databases, beside some homology with the hypothetical “capsid” protein of a few partitiviruses. The dsRNA3 encodes a protein with no similarity to any protein in the GenBank database. SDS-PAGE and polypeptide mass fingerprint-mass spectrum (PMF-MS) analyses indicated that the CP of the AfPV1 was encoded by dsRNA2. Phylogenetic analysis showed that the AfPV1 and relative viruses were found in an unclassified group inside the Partitiviridae family. AfPV1 seems to result in debilitation symptoms, but had no significant effects to murine pathogenicity. These findings provide new insights into the partitiviruses taxonomy and the interactions between viruses and A. flavus.The opportunistic human pathogenic fungus  Partitiviridae are classified into four genera, namely Alphapartitivirus,Betapartitivirus,Gammapartitivirus, and Deltapartitivirus  image reconstruction, as well as one determined by X-ray crystallography , causing obvious symptoms on its host morphology was found. The influence of AfPV1 on pathogenicity of A. flavus generating virus-infected and virus-free isogenic lines and the unique characteristics of AfPV1 were analyzed and discussed here.With the increasing discovery of mycoviruses that are able to selectively infect human pathogenic fungi, mycoviruses have shown the significant potential for biological control agents . So it ssymptoms . Mycovirsymptoms . In ordeA. flavus LD-3-8 was isolated from patient’s room of Affiliated Hospital of Guizhou Medical University. A virus-free isolate LD-F was obtained from isolate LD-3-8 by single asexual spore isolates. Isolate LD-F1 derived from LD-F, was labeled with a pyrithiamine resistance (ptr) gene using PEG-mediated methods. All A. flavus isolates were cultured on potato dextrose agar (PDA) plates and Czapek Agar (CZ) plate at 30°C and stored on PDA slants at 4°C.The single-spore isolate A. flavus were cultured on sterilized cellophane films placed on PDA plates (9 cm in diameter) at 30°C in the dark for 3–5 days. Mycelia in each plate were harvested and stored at -80°C until use. Extraction and purification of the dsRNAs from mycelia was carried out using CF-11 cellulose  column chromatography as previously described  amplification method, sequencing of the cDNAs, and analysis of the sequences were done using the procedures described in our previous studies  extracted from isolate LD-3-8 of  studies . The ter studies .The full-length cDNA sequence for dsRNA1, dsRNA2, or dsRNA3 was obtained from assembling the partial sequences in the different cDNA clones. ORFs in full-length cDNA sequence were searched using DNAMAN 7.0 . Amino acid sequences were aligned using Clustal W implemented in MEGA 6.06 . The phyA. flavus isolate LD-3-8 using the modified method described previously (g for 30 min). The supernatant was then subjected to ultracentrifugation at 120,000 × g for 2 h under 4°C to precipitate the virus particles in the supernatant. The supernatant containing the virus particles was then overlaid on a centrifuge tube containing sucrose density gradient (10–40%) and centrifuged at 70,000 ×g for 3 h at 4°C. The gradient containing VLPs was diluted with PBS and ultracentrifuged at 120,000 ×g for 2 h at 4°C. Five separated fractions were individually measured for the presence of the virus particles by dsRNA detection using the agarose gel electrophoresis. The fractions containing virus particles were carefully collected and suspended using 200 μl PBS. The virus particles were observed under transmission electron microscopy (TEM) after staining with 2% (wt/vol) phosphotungstic acid solution (pH 7.4).The viral particles (VLPs) were isolated from eviously . The isoThe purified virus particles suspension was loaded on a sodium dodecyl sulfate (SDS)-polyacrylamide (12%) gel stained with Coomassie brilliant blue R250. The resulting protein bands on the gel were individually excised and subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) analysis at Sangon Biotech . To avoid missing any proteins during virion preparation, purified virus particles suspension was also directly subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis at Sangon Biotech .ptr gene using PEG-mediated methods as previously described (ptr isolate (named LD-F1) with no observable differences in biological properties compared to the parental isolate was used in the paired-culture method as previously described  from the margin of each colony was inoculated onto the center of PDA and CZ plates (9 cm in diameter), and cultured at 30°C in the dark. The radial growth rate (RGR) was determined for each developing colony  at 2 days intervals over a week period as follows: RGR (cm/d) = [(D 4-D 2)/2]/2, where D 4 and D 2 represent the diameter of 4 and 2 days old colonies in each plate, respectively.A. flavus were harvested in 2 mL saline buffer, and spore numbers for each colony were estimated using a haemocytometer; the data were transformed to log 10 before analyses. The length of spore chains were measured under microscope.After 6 days incubation in the dark at 30°C on PDA plates (9 cm in diameter), conidiospores of A. flavus burden quantitatively, quantitative PCR (qPCR) was tested on total genomic DNA extracted from homogenized whole lungs of mice using a previously described method  to amplify A. flavusβ-tubulin (M38265), and the specific primers (Muact-f:GAGACCTTCAACACCCCAGC and Muact-r: ATGTCACGCACGATTTCCC) to amplify the murine actin (NM_007393) as an internal control. Quantification of both gene targets was performed in triplicate, using ten independent infected mice, and saline-injected mice as control. qPCR assay was performed using SYBR green I . The data of threshold cycle (Ct) were analyzed with unpaired t-tests using GraphPad Prism 5.01 . In order to confirm this model represent the virulence of the fungus A. flavus, microscopic and histological changes of mice lung tissues were observed after infection using a previously described method  were detected in single spore isolate LD-3-8 . The fulPartitiviridae, including Botryosphaeria dothidea virus 1 , Colletotrichum acutatum RNA virus 1 , and Valsa cypri partitivirus   . No othe virus 1 . MoreoveThe 5′-untranslated regions (UTRs) of the plus strands of dsRNA1, dsRNA2 and dsRNA3 are 18, 112, and 124 bp long, respectively, and show conserved nucleotides at the 5′ terminus (AAACUUUUGU) . A similA. flavus isolates. In our study, dsRNA1, dsRNA2, and dsRNA3 could not be detected separately and they always appear to coexist in A. flavus. Moreover, the three segments appear to coexist in purified VLPs , matched to the deduced amino acid sequence of the protein encoded by dsRNA2 (p < 0.05), matched to the deduced amino acid sequence of the protein encoded by dsRNA2  and a new genus (Epsilonpartitivirus) in family of Partitiviridae  were found in a unclassified cluster, and this cluster grouped clearly outside of the five genera in Partitiviridae family  infected mice appeared large numbers of bleeding area compared to saline-injected mice, but no significant differences between LD-F1 and LD-F1-b infected mice (A. flavus was detected in mice lungs tissue of A. flavus (isolate LD-F1 and LD-F1-b) infection, but was not detected in saline-injected mice , the larger one encodes RNA-dependent RNA polymerase (RdRp) and the smaller one encodes CP . These results indicates the CPs of the viruses in this group may be new CP category in family Partitiviridae. AfPV1 and Botryosphaeria dothidea virus 1 contains three segments in each genome, but C. acutatum RNA virus 1, U. virens partitivirus 2 and U. virens partitivirus 3 possesses two segments in each genome, that is the difference in this group  caused abnormal colony phenotypes, slow growth, and production of light pigmentation on its host A. fumigatus (Members of the family otential . Howeverumigatus . In our umigatus .Partitiviridae family for which we propose the name Zetapartitivirus. Horizontal transmission analysis revealed that AfPV1 infection causes debilitation symptoms but no significant alterations to murine pathogenicity of A. flavus.In conclusion, we have isolated and characterized a new partitivirus with 3 genome segments and provided convincing evidence that dsRNA2 encodes a CP, whereas the function of the ORF encoded by dsRNA3 remains unknown. Phylogenetic analysis suggest that AfPV1 belongs to a new genus inside the YJ designed and analyzed the data. JW collected and analyzed the data. BY purified the virus particles. JZ analyzed the mice aspergillosis model. QW and WY gave some advises for the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cryptococcus neoformans and Cryptococcus gattii isolates from 60 renal transplant recipients in Brazil were characterized. Clinical characteristics of the patients and prognostic factors were analysed. Seventy-two (87.8%) isolates were C. neoformans and 10 (12.2%) were C. gattii. VNI was the most common molecular type , followed by VNII , VGII , VNB  and VNI/II . The isolates showed a high genetic diversity in the haplotype network and six new sequence types were described, most of them for VNB. There was a bias towards skin involvement in the non-VNI population (P = .012). VGII isolates exhibited higher fluconazole minimum inhibitory concentrations compared to C. neoformans isolates (P = 0.008). The 30-day mortality rate was 38.3%, and it was significantly associated with fungemia and absence of headache. Patients infected with VGII had a high mortality rate at 90 days (66.7%). A variety of molecular types produce disease in renal transplant recipients in Brazil and highlighted by VGII and VNB. We report the clinical appearance and impact of the molecular type, fluconazole susceptibility of the isolates, and clinical characteristics on patient outcome in this population.Genotypic diversity and fluconazole susceptibility of 82  Cryptococcus neoformans and Cryptococcus gattii [C. neoformans has a worldwide distribution affecting predominantly individuals with impaired cell-mediated immunity and C. gattii has a more limited environmental distribution and a higher percentage of disease within apparently normal hosts [Cryptococcosis is a life-threatening invasive fungal disease caused by the encapsulated yeasts, s gattii . C. neofal hosts . In renaal hosts .C. neoformans/C. gattii species complexes is continuing to evolve under molecular evidences [C. neoformans and C. gattii and currently these species can be further divided into ten molecular siblings known as VNI, VNII, VNB, VNIII, VNIV, VGI, VGII, VGIII and VGIV [The nomenclature of vidences ,13. Howeand VGIV  with a pand VGIV ,15.C. neoformans and C. gattii species complexes in the transplant recipient relies on small series and case report [Clinical comparative studies and understandings between different cryptococcal molecular types are still in their infancy and remain controversial whether or not these different molecular types represent specific characteristics in terms of clinical manifestations or attributable mortality rates ,16–18. Fe report ,19–23.C. neoformans and C. gattii isolated and to assess the clinical outcome of cryptococcosis and their molecular types in patients undergoing renal transplantation throughout Brazil. Interestingly, Brazil represents an environment with a diverse number of cryptococcal molecular types and likely has the most cryptococcal strain diversity of any country practising routine kidney transplantation [The purpose of our study was to characterize the molecular types of antation ,25.We enrolled a total of 60 renal transplant recipients followed for a median period of 4 months (0 days to 11 years). One patient had received a liver transplant allograft one year before kidney transplantation and another patient had undergone simultaneous pancreas-kidney transplantation. The clinical characteristics are outlined in C. neoformans/C. gattii species complexes from 60 renal transplant recipients. Ten (12.2%) isolates were identified as C. gattii and 72 (87.8%) isolates as C. neoformans (We collected 82 isolates of URA5 locus (1 new), 10 for LAC1 (1 new), 9 SOD1 (1 new), 14 for IGS1 region (3 new), 10 for CAP59 (1 new), 11 for PLB1 and 8 for GPD1 (1 new). Based on the combined analysis of the 7 MLST loci for C. neoformans, 20 sequence types (ST) were found, in which 6 were new . Of those new ST, four were VNB molecular type and two VNII (Supplementary Table 1).A total of 81 isolates were analysed by MLST and compared to MLST database. Nine allele types have been identified for the C. neoformans/C. gattii species complexes, being 81 generated in this study and 63 recovered from GenBank (Supplementary Table 1). Haplotype and nucleotide diversities were high in our overall dataset  and also specifically within our 81 transplant recipient isolates . Remarkably, 24 out of 71 different haplotypes found in the full dataset for C. neoformans/C. gattii species complexes originated from transplant recipients. Only four haplotypes  were identical to reference strains, revealing the high diversity of our isolates  from C. gattii were more likely to be a retransplant than those infected with C. neoformans (P = .046) (P = .012).Regarding clinical correlations, patients infected by  = .046) . FurtherC. neoformans and C. gattii isolates varied as depicted in −1, with the higher mode for VGII (16 mg l−1) and lowest mode for VNII (2 mg l−1). For example, 13 out of 82 (15.85%) isolates exhibited fluconazole MIC values that were ≥16 mg l−1, in which 8 (61.54%) were represented by molecular type VGII  (Fluconazole minimum inhibitory concentration (MIC) distributions for  = .008) .Table 3−1. After multivariate regression analysis, the factors independently associated with 30-day mortality were fungemia  and absence of headache    died within 30 days after diagnosis of cryptococcosis, and 4 more  died within 90 days. Finally, the overall mortality rate in our study group was 61.7% (37/60), over the entire follow-up period. Based on the univariate analysis, the following factors were significantly associated with high mortality at 30 days: induction immunosuppressive therapy, deceased donor, receipt of a calcineurin-inhibitor agent, pulmonary infection, fungemia, somnolence and absence of headache at admission, CSF antigen titre >1:512 at diagnosis, positive pulmonary culture, discontinuation of tacrolimus after infection, graft loss within 30 days and patients infected with isolates showing MIC ≥ 16 mg l = .001) . Most ofnk test) .Figure C. gattii, there was an impressive 66.7% mortality rate within 90 days. Similarly, other investigators have found high rates of dissemination (63.6%) and a high 90-day mortality (36%) for solid organ transplant (SOT) recipients infected by C. gattii [Cryptococcus in an individual transplant recipient infection for prognostic determination and possibly management alterations.Our retrospective analysis describes 60 cases of cryptococcosis among renal transplant recipients documented during a period of 26 years. The patients presented with high rates of fungemia 38.3%), common disseminated infection (63.3%), and substantial 90-day mortality (45%). These data are comparable to some prior studies evaluating cryptococcosis in renal transplant recipients 8.3%, com,26,27. H. gattii . These oWe observed clinically that fungemia and absence of headache were significantly correlated with decreased survival in our kidney transplant recipients. These characteristics are likely to identify a patient with a high burden of yeasts and/or late diagnosis. These clinical characteristics have been found to be poor prognostic features in other patient populations ,30.C. neoformans/C. gattii species complexes and even a location for species origin [Cryptococcus infecting transplant recipients remain scarce worldwide. In the setting of SOT, one study in China revealed 9 cases of VNI and a recent study described 10 strains of molecular type VGII and one of VGI from the Pacific Northwest of USA outbreak. In this last study, 18% of the strains in transplant recipients were C. gattii [C. gattii represented 12% of isolates (VGII). While VNI was the most common molecular type infecting our patients (66.7%), some genotypic diversity with C. neoformans was also found as we detected 14 VNII and 6 VNB isolates. The molecular predominance of VGII may represent either a specific tropism for transplant recipients and/or increased resistance to tacrolimus/cyclosporine or more likely higher exposure to VGII in the environment over the other VG molecular types. A higher frequency of C. gattii disease observed in retransplantation recipients supports the hypothesis that cryptococcal disease of immunocompromised hosts with C. gattii may be more a primary disease while C. neoformans disease more frequently represents secondary or reactivation disease [In Brazil, most cryptococcal molecular types in infections are represented. Indeed, MLST and whole-genome based population analyses suggest that Brazil could be a global centre for diversity of s origin ,25,31–33. gattii . We founC. neoformans in this Brazilian cohort, a molecular type that was considered to be geographically restricted to Africa [Surprisingly we found VNB isolates of o Africa . Howevero Africa ,35–39. Io Africa . This iso Africa .GPD1 locus for VGII molecular type as previously reported [The Next Generation MLST (NGMLST) methodology and primers that we used were not able to properly sequence the reported . Anotherreported ,33,41.C. neoformans and C. gattii infections with the important caveat that there were fewer C. gattii strains and all were VGII. Consequently, the statistical power to detect differences in our study is limited. However, these initial observations support the hypothesis that virulence is not consistently associated with a single major molecular type or subtype but may be more related to the distinct properties of individual isolates.The clinical impact of different cryptococcal molecular types on patient outcome is still unknown. Controversial results have been generated in the correlation between molecular types and virulence based on experimental studies ,16,18 anC. neoformans/C. gattii species complex isolates exhibiting high fluconazole MICs did correlate with a worse patient survival rate. However, the precise clinical correlation of in vitro antifungal susceptibility testing of Cryptococcus and break points remains to be further defined [Fluconazole susceptibility profile is a potentially important clinical issue but its relevance in terms of precisely determining prognosis is still controversial, especially in the setting of the transplant population ,19,44,45 defined .In conclusion, cryptococcosis in the setting of SOT in less-resourced countries should be considered a life-threatening fungal disease with substantial mortality rates. The high mortality is probably due to late diagnosis, suboptimal antifungal therapy, including lack of 5FC, as well as the virulence properties of these fungi in SOT. In fact, this transplant population in Brazil represents a relatively uniform host with high rates of mortality for cryptococcosis that may allow for the detection and characterization of particularly virulent strains. With this diversity of strains and a similar high-risk host population for mortality, further examination of these transplant cohorts in Brazil and their fungal strains may give us signals into how these yeasts specifically cause aggressive disease.C. neoformans and C. gattii strains isolated from patients who underwent renal transplantation and subsequently developed cryptococcosis from 1987 to 2013 at São Paulo Hospital or the Kidney Hospital in São Paulo. The isolates were stored in cryopreservative medium at −70°C with Yeast Peptone Dextrose (YPD) and 20% glycerol at the Special Mycology Laboratory – Federal University of São Paulo.We evaluated all The following variables were collected from the medical records using a standard clinical report form: demographic data, complications prior to and after transplantation, immunosuppressive regimens, antifungal therapy, and laboratory data. Disseminated cryptococcosis was defined as the involvement of at least two noncontiguous organ systems or the presence of fungemia . PrimaryCAP59, LAC1, PLB1, SOD1, URA5, TEF1, GPD1) and the intergenic spacer region 1 (IGS1) of the nuclear ribosomal RNA gene. The primer sequences and the preparation of the sequencing libraries were based on the NGMLST method previously described and the raw sequencing data was processed using MLSTEZ [http://mlst.mycologylab.org).A single colony of each isolate was isolated, grown in YPD broth and frozen at −70°C with glycerol. To isolate genomic DNA, the isolates were streaked from these frozen stocks onto fresh YPD agar, grown for 2–4 days. Next, several colonies of each sample were used to extract and purify the genomic DNA, using the MasterPure Yeast DNA Purification Kit . We analysed the sequences from eight loci, including seven standard gene regions . A value of P ≤ .05 was considered statistically significant.Continuous data were presented as either the mean ± SD or the median and range, and categorical data were presented as proportions. Univariate analyses were performed to compare categorical variables using either the Chi-square or Fisher's exact test as appropriate and continuous variables by Student's http://mlst.mycologylab.org) and GenBank (https://www.ncbi.nlm.nih.gov/genbank/).The sequences of all newly identified allele types have been submitted to the MLST database (


Natural products from plants, such as chemopreventive agents, attract huge attention because of their low toxicity and high specificity. The rational drug design in combination with structure-based modeling and rapid screening methods offer significant potential for identifying and developing lead anticancer molecules. Thus, the molecular docking method plays an important role in screening a large set of molecules based on their free binding energies and proposes structural hypotheses of how the molecules can inhibit the target. Several peptide-based therapeutics have been developed to combat several health disorders, including cancers, metabolic disorders, heart-related diseases, and infectious diseases. Despite the discovery of hundreds of such therapeutic peptides however, only few peptide-based drugs have made it to the market. Moreover, the in silico activities of cyclic peptides towards molecular targets, such as protein kinases, proteases, and apoptosis related proteins have not been extensively investigated. In this study, we explored the in silico kinase and protease inhibitor potentials of cyclosaplin, and studied the interactions of cyclosaplin with other apoptosis-related proteins. Previously, the structure of cyclosaplin was elucidated by molecular modeling associated with dynamics that were used in the current study as well. Docking studies showed strong affinity of cyclosaplin towards cancer-related proteins. The binding affinity closer to 10 kcal/mol indicated efficient binding. Cyclosaplin showed strong binding affinities towards protein kinases such as EGFR, VEGFR2, PKB, and p38, indicating its potential role in protein kinase inhibition. Moreover, it displayed strong binding affinity to apoptosis-related proteins and revealed the possible role of cyclosaplin in apoptotic cell death. The protein–ligand interactions using LigPlot displayed some similar interactions between cyclosaplin and peptide-based ligands, especially in case of protein kinases and a few apoptosis related proteins. Thus, the in silico analyses gave the insights of cyclosaplin being a potential apoptosis inducer and protein kinase inhibitor. Cancer is a well-recognized global health problem responsible for ∼7.6 million deaths  worldwide, which is expected to rise to 13.1 million by 2030 . Despite the advancements in the field of cancer research, there is still an urgency to discover and develop anti-cancer therapeutics. Natural products are of particular interest as chemopreventive agents because of their low toxicities and potential efficacies . RecentlACD/ChemSketch 12.01, AutoDock Vina 1.1.2, Avogadro, CycloPsWeb, GROMACS, LigPlot, Modeller 9.2, MGL tools, Open Babel, Protein Data Bank (PDB), PubChem, PyMOL, and Swiss Target Prediction.The cyclic octapeptide (cyclosaplin) and various peptides (positive control) for specific proteins were used as ligands for docking studies . The pepoMolecular mass less than 500 Da;oHigh lipophilicity (expressed as LogP less than 5);oLess than 5 hydrogen bond donors;oLess than 10 hydrogen bond acceptors;oMolar refractivity between 40 and 130.The peptide based-ligand molecules selected for docking experiments were screened for Lipinski’s rule of five. Lipinski’s rule of five  states thttp://www.scfbio-iitd.res.in/utility/LipinskiFilters.jsp).Lipinski’s rule of five was also checked in Supercomputing Facility for Bioinformatics and Computational Biology, IIT Delhi, wherein PDB structures of the molecules were uploaded to the online server  were docked with target proteins using AutoDock Vina 1.1.2 . The recLigPlot was used to study protein–ligand interactions for a given pdb file encrypting the docking . The LigThe ligand structures were drawn in CycloPsWeb or downloaded from PubChem and converted to pdb format using Open Babel and modeled using GROMACS and MODELLER 9.2 program along with cyclosaplin . The strThe ligands prepared for docking were screened for Lipinski’s rule of five. The commercially available or reported peptide inhibitors/inducers (positive control) were also tested against the target proteins respectively .Swiss Target Prediction was used to predict the possible targets of cyclosaplin a. A listThe docking process was carried out using AutoDock Vina. The docking scores are graphically represented in The protein–ligand interaction study was performed using LigPlot. The interactions of the ligands cyclosaplin and various peptide-based ligands with amino acids residues of the target proteins are shown in Peptides are effective receptor-binding ligands; many other classes of ligands sharing this binding trait include small molecules, endogenous proteins, and antibodies . Cyclic Santalum album L. [In our previous study, cyclosaplin was isolated, purified, and characterized from album L. . The cycalbum L. . In spit50 2.06 µg/mL in MDA-MB-231 cells .The potential targets of cyclosaplin were predicted by Swiss Target Prediction  Figure a and theIn contrast to most small molecule drugs, peptides have high affinity, strong specificity for targets, and low toxicity, whereas, in contrast to chemotherapeutics antibodies, they have good penetration of tissues because of their small size ,34,35,36The interaction of the cyclosaplin and other peptide-based ligands (positive control) with the amino acids of various cancer-related proteins were also determined . We prevSome of the amino acids of MMP-2 interacted with both the peptide ligands (CTTHWGFTLC and cyclosaplin), forming H-bonds and hydrophobic contacts F. SimilaIn caspase 9 I, Gly276Previously, in a 3D cell culture, the efficacy of cyclosaplin was shown as an anticancer agent . Apart fThe structure of the cyclic octapeptide was elucidated previously by molecular modeling associated with dynamics and was used in the docking studies. Docking studies showed the strong affinity of cyclosaplin towards cancer-related proteins, especially protein kinases and apoptosis-related proteins. Thus, the in silico analyses revealed the potential of cyclosaplin as an apoptosis inducer and a protein kinase inhibitor. Based on these studies, appropriate in vitro and in vivo experiments should be designed rationally to validate its biological activity.