Some observational studies and randomized controlled trials (RCTs) have suggested an association between abacavir (ABC) use and myocardial infarction (MI), whereas others have not.This pooled analysis of 66 phase II–IV RCTs estimates exposure-adjusted incidence rates (IRs) and relative rates (RRs) of MI and cardiovascular events (CVEs) in participants receiving ABC- and non-ABC-containing combination antiretroviral therapy (cART). The primary analysis of MI included ABC-randomized trials with ≥48-week follow-up. Sensitivity analyses of MI and CVEs included non-ABC-randomized and <48-week follow-up trials.In 66 clinical trials, 13 119 adults  were on ABC-containing cART and 7350 were not. Exposure-adjusted IR for MI was 1.5 per 1000 person-years  in the ABC-exposed group and 2.18 per 1000 PY  in the unexposed group. The IR for CVEs was 2.9 per 1000 PY  in the exposed group and 4.69 per 1000 PY  in the unexposed group with studies of ≥48 weeks of follow-up, with an RR of 0.62 . The inclusion of nonrandomized and shorter-duration trials did not significantly change the RR for MI or coronary artery disease.This pooled analysis found comparable IRs for MI and CVEs among ABC-exposed and -unexposed participants, suggesting no increased risk for MI or CVEs following ABC exposure in a clinical trial population. Modifiable risk factors for MI and CVEs should be addressed when prescribing ART. Cardiovascular disease (CVD) is a leading cause of death in HIV-positive individuals, accounting for approximately 11% of the total deaths in this population . The risSeveral studies have suggested an association between abacavir (ABC) use and increased risk of MI , 14–19, Since the last analysis of GSK/ViiV Healthcare clinical trial data in 2009 [Studies were identified through the GSK clinical trial repository. This pooled analysis was based on study reports from all GSK/ViiV Healthcare–sponsored clinical trials in which participants were exposed to ABC, with ≥24 weeks of exposure to cART and for which at least the primary objective had been completed by December 2016. Exposure was defined as being on an ABC-containing regimen or non-ABC-containing regimen as part of study randomization or standard of care. Antiretroviral therapy (ART) taken before study entry was not considered. Each study participant contributed to only 1 of the exposure categories, except in the STRIIVING study, in which participants were allowed to switch after the initial treatment. Study-level data were assessed if participants were randomized to receive ABC. Participant-level data were used if ABC was assigned as standard of care at the discretion of the investigators.The same definitions as in the initial meta-analysis in 2009 by Brothers et al. [The following specific preferred terms were used for MI: acute myocardial infarction and myocardial infarction. The following specific preferred terms were used for CVE: arteriosclerosis coronary artery, coronary artery disease, coronary artery occlusion, angina pectoris, angina unstable, acute myocardial infarction, myocardial infarction, and myocardial ischemia.Any preexisting events were not collected as part of the clinical trials unless there was a change in severity during the trial. Therefore, any pretrial MIs and CVEs were not collected and not included in the database.All statistical analyses were performed using SAS software, version 9.3 . The number of participants, exposure to ABC- or non-ABC-containing regimens in person-years, and number of events were calculated for all studies and summarized according to the exposure category (ABC vs non-ABC). For studies in which participants were randomized to ABC or non-ABC regimens, mean exposure was determined on study level, as opposed to participant-level exposure in trials where ABC was assigned at the investigators’ discretion. The total number of exposed days in each trial was divided by 365.25 to obtain the total exposure in person-years.Proportions of MIs and CVEs were calculated with their exact binomial 2-sided 95% CIs. A Poisson regression model was fitted to estimate incidence and relative rates of MI and CVEs, which were expressed as rates per 1000 person-years with 95% CIs. The log link was used with an offset, because rates rather than number of events were being modeled. To account for the discontinuation of ABC-containing or non-ABC-containing regimens, incidence rates per 1000 person-years were adjusted for the time participants were exposed to ART during the study period . There was no adjustment for confounders. The same summary measures are reported for the main and all sensitivity analyses.The main analysis included data from studies that were randomized to ABC or non-ABC-containing regimens and included follow-up of ≥48 weeks; 12 studies were included in the previous meta-analysis , and 5 Grandomized to ABC or control studies with follow-up durations of <48 weeks conducted for MIs;randomized or nonrandomized to ABC or control studies with follow-up durations of ≥48 weeks conducted for MIs and CVEs;randomized or nonrandomized studies with follow-up durations of <48 or ≥48 weeks conducted for MIs and CVEs.Only studies in which individuals were exposed to ABC-containing cART were eligible for inclusion. A total of 14 studies conducted since the last meta-analysis  were incThe age range of participants across studies was 18 to 85 years. The majority of study participants were male across studies, except for ARIA in which all participants were female. Overall, for ABC-exposed and ABC-unexposed participants, the exposure-adjusted incidence rates of MI and CVEs ranged between 1.46 and 4.65 per 1000 person-years across the different analyses in this study, and they are largely in line with—although slightly lower than—previously reported rates in HIV-positive individuals. Although the MI incidence rates were slightly higher among ABC-unexposed participants, the CIs overlapped with those in ABC-exposed participants, indicating no differences observed between the groups. This result is similar to the conclusion in previous GSK meta-analyses . The incAccording to the Centers for Disease Control and Prevention, 76% of all HIV-positive adults and adolescents in the United States in 2010 were male , which iOnly a review of the GSK/ViiV Healthcare clinical trial database was performed to identify studies for inclusion. It is possible that published data from non–GSK/ViiV Healthcare–sponsored clinical trials might have provided additional insights. As the clinical trials were not specifically designed to evaluate cardiovascular outcomes, the collection of baseline risk factors for CVEs may not have been incorporated in the original study protocols. Although there was only limited variation in the available cardiovascular risk factors at study entry, cardiac events were likely a stopping criterion in the clinical trials, and therefore none of the trial participants had multiple cardiac events. Additionally, there was no additional adjudication for MI and CVE for the current study. Furthermore, pretreatment events were not recorded, making it impossible to determine whether the event reported during the clinical trials was a recurrence or a new event. Although most trials only included treatment-naïve participants, the definition of treatment naïve varied from not having been on any ART for more than several weeks since diagnosis to not having been on specific ART classes before. It is possible that these other ART exposures may have influenced the risk of CVEs as well , but it Based on these limitations of the available data, the current pooled analysis is mainly descriptive in nature, and the results might only be generalizable to clinical trial populations. However, precautionary text regarding CVE risk is included in ABC-related product leaflets, possibly making it more likely that the number of cardiac events outside of clinical trials is limited due to lifestyle advice to patients and potentially fewer ABC prescriptions to patients at high risk for CVEs.MIs and CVEs were uncommon in GSK/ViiV Healthcare–sponsored clinical trials in which participants were exposed to ABC-containing cART. This analysis found comparable incidence rates for MI and CVEs among ABC-exposed and -unexposed participants, suggesting no increased risk for MI or a CVE following ABC exposure in a clinical trial population. This updated analysis, which includes an additional 3999 person-years of ABC exposure and only 3 additional MIs, continues to corroborate the results from previous meta-analyses of clinical trial data , 21. How


Miscommunication during inter-unit handoffs between emergency and internal medicine physicians may jeopardize patient safety. Our goal was to evaluate the impact of a structured communication strategy on the quality of admission handoffs.We conducted a mixed-methods, pre-test/post-test study at a 560-bed academic health center with 60,000 emergency department (ED) patient visits per year. Admission-handoff best practices were integrated into a modified SBAR format, resulting in the Situation, Background, Assessment, Responsibilities & Risk, Discussion & Disposition, Read-back & Record (SBAR-DR) model. Physician handoff conversations were recorded and transcribed for the 60 days before (n=110) and 60 days after (n=110) introduction of the SBAR-DR strategy. Transcriptions were scored by two blinded physicians using a 16-item scoring instrument. The primary outcome was the composite handoff quality score. We assessed physician perceptions via a post-intervention survey.The composite quality score improved in the post-intervention phase . Three of the 16 individual scoring elements also improved, including time for questions  and confirmation of disposition plan . The majority of emergency and internal medicine physicians felt that the SBAR-DR model had a positive impact on patient safety and handoff efficiency.Implementation of the SBAR-DR strategy resulted in improved verbal handoff quality. Agreement upon a clear disposition plan was the most improved element, which is of great importance in delineating responsibility of care and streamlining ED throughput. Future efforts should focus on nurturing broader physician buy-in to facilitate institution-wide implementation. Patient care handoffs are a potentially vulnerable time for patient safety.13Standardized handoff communication tools have been shown to improve outcomes for inter-unit handoffs,20In 2012 Beach et. al. published best practice recommendations for ED-to-inpatient handoff communication, including style, form and content. They suggested synchronous, two-way, closed-loop communication, with the goal of constructing a shared mental model of patient care between EPs and IM providers. Rather than rote recitation of data, it was suggested the content of handoff communication should focus on clinical judgment, diagnostic uncertainty, the patient’s clinical trajectory, pending tasks, and any patient- or system-level considerations that may impact care.21A study in 2016 found that a standardized method of handoff for patients admitted to a geographically isolated, 35-bed community hospital from the ED resulted in fewer physician-reported “defective” handoffs;We conducted a mixed-methods, pre/post-study of admission handoff quality in conjunction with the implementation of the SBAR-DR strategy for admission handoff communication between EPs and IM physicians. The intervention took place at a 560-bed, Midwestern academic health center. The ED is a certified Level I trauma center with 60,000 patient visits per year, 15,000 inpatient admissions per year, and an emergency medicine (EM) residency program with 27 trainees. The IM service includes teaching and non-teaching teams, which admit approximately 6,000 patients per year, 60% of which are through the ED. The IM residency program has approximately 80 house officers. Prior to the intervention, there was no institutional standardized verbal or written handoff strategy. The project team consisted of resident and faculty physicians from EM and IM, as well as an educational expert specializing in training and performance improvement.What do we already know about this issue?Inter-unit handoff from the ED to the inpatient setting is a vulnerable time for patient safety, but little research has investigated strategies to improve this process.What was the research question?How would a structured communication strategy impact the quality of handoffs between emergency physicians and internal medicine physicians?What was the major finding of the study?The admission handoff quality score improved following the intervention .How does this improve population health?Improving handoff practices has the potential to improve the care of the 12 million people/year admitted to the hospital from the ED.The research team conducted iterative rounds of analysis to integrate admission handoff best practicesAn admission handoff template was created within the EHR to supplement verbal handoff. The template was designed as an editable macro that could quickly be imported into the EP note. The template included headings for working diagnosis, description of the ED course, and a drop-down menu to list potential risks to patient care . Data on pending tests and medications administered were automatically imported into the template. A definitive assignment of care responsibilities was documented at the end of the note, along with pager numbers for the appropriate admitting service to facilitate communication with ED nursing and ancillary staff. The EP completed the handoff note immediately after completion of the verbal admission handoff.The research team developed an educational session that included a discussion of admission handoff best practices, a review of internal handoff data, and introduction to the verbal and written elements of the SBAR-DR model. The training included review and group discussion of two videos, one demonstrating poor handoff communication and one demonstrating high-quality communication using SBAR-DR.Admission handoff conversations were recorded from two labeled ED telephone lines using a HIPAA-compliant online recording program for 60 days prior to  and 60 days following  the implementation of the SBAR-DR process. Participants were emailed consent cover letters prior to the start of the intervention to notify them that their calls could be recorded.Calls from the recorded phone lines were initially screened based on length of call and excluded if less than 25 seconds. The remaining calls were reviewed three times by a member of the research team (RB), and were excluded if they did not concern an inpatient admission, involved admitting services other than IM, or the screener deemed the recording quality did not allow accurate evaluation . Eligible calls underwent stratified random sampling to achieve the pre-determined sample size. (See below.) We used sample stratification to ensure that the distribution of EP training level within the cohort of eligible calls was similar to the distribution in the final pre-/post-intervention samples. Recordings were de-identified and transcribed verbatim by a hospital-approved, independent third party.We created a 16-point scoring instrument reflecting the best practice recommendations used in creating SBAR-DR.Admission handoff transcripts were randomly assigned and independently scored by one of three dyads. Each of the dyads were comprised of one EP and one IM physician of similar training level to minimize the potential for undue influence. The dyads were blinded to physician- and patient-identifying information and whether the transcription was from the pre- or post-intervention group. Scoring disagreements were settled by consensus via in-person conference of dyad members.The primary outcome was the composite admission handoff score (0–16 points), which was determined by summing the “communicated” elements of the transcribed verbal handoffs. Secondary outcomes included frequency of individual handoff elements; a global rating based on an anchored, five-point scale; and average length of handoff calls.We developed a post-intervention survey to assess EPs’ and IM physicians’ perceptions of the SBAR-DR strategy. Questions focused on patient safety and efficiency using a five-point, Likert-like scale. Before distribution, the survey was pilot tested for clarity and face-validity by two EPs and three IM physicians. A consent cover letter and link to an anonymous online survey was sent to eligible participants via their university email accounts. Participants who reported they had not participated in an ED admission handoff during the study period were excluded from the analysis.Unpublished pilot data demonstrated that handoff scoring elements were communicated 30% of time. Anticipating a 25% absolute improvement,We compared mean composite admission handoff quality scores and global rating scores using a t-test. Individual scoring elements were compared using chi-square tests. We calculated percent agreement and kappa statistics to determine inter-rater reliability for scoring elements. For the composite quality score and global rating scale, a general linear model was fit that included fixed-effect terms for time period (pre- or post-), EP training level, and the interaction of time period by training level. Type III tests were performed and, if significant, were followed by analysis of all possible pairwise comparisons of interest.We calculated descriptive statistics for physician survey responses and handoff template use within the EHR. The method in which the written note was coded did not allow for analysis beyond descriptive terms and only produced data in the form of general use counts. Statistical calculations were completed using IBM SPSS v 22 and PC SAS version 9.4. We considered p-values of <0.05 statistically significant. This research project was approved for exempt status by the local institutional review board (#729-14-EX).Approximately 14,400 calls took place on the recorded phone lines over the study period, with 20% lasting >25 seconds . After rThe inter-rater agreement for SBAR-DR scoring elements was moderate (0.41–0.60) to substantial (0.61–0.80) as measured by Cohen’s kappa.The post-intervention survey response rate was 50% for EPs (19/38) and 66% for IM physicians (20/30). We found that the introduction of a standardized handoff process for patients being admitted from the ED to hospital setting resulted in improvements in verbal handoff quality. The driver of improvement was primarily due to improvements in opportunities for questions and reaching unambiguous agreement regarding patient disposition. Interactive questioning during handoffs is recommended by regulatory agencies28Explicit agreement in disposition plan was also an important improvement. Uncertain assignment of responsibility is a known barrier to safe care transitions.As a result of using the SBAR-DR strategy, over half of surveyed physicians reported personally experiencing improved patient safety during the 60-day study period. Although the absolute improvement in quality scores was modest (approximately 12% improvement above baseline), the intervention resulted in the communication of approximately 100 pieces of additional information, any of which had the potential to improve the handoff process.A recent survey of EM residency programs in the U.S. found poor adherence to standardized ED-to-inpatient handoff practices,30Additionally, handoff practices are an engrained part of a specialty’s culture. Although our study group included faculty and resident physician champions from IM and EM, we may not have fostered adequate buy-in from practicing providers to change practice routines. As institutions implement changes to inter-unit handoffs and care transitions, they need to address cultural complacency and build coalitions among affected members of the healthcare team.32The study had several limitations. Implementation was conducted at a single institution, so results may not be generalizable to other settings. The pre/post study design cannot exclude the possibility that factors other than the intervention may have influenced the results. Since we scored written transcripts, we may have missed certain cues, such as voice inflection and tone, which can be important in verbal communication. Additionally, we used a novel scoring instrument, as we were unable to find a published, psychometrically-tested assessment instrument. Our scoring system was strict in its definitions of “communicated,” which may have biased results toward the null. Finally, the method in which the written note was coded in the EHR did not allow for analysis beyond descriptive counts.We found that introduction of a standardized admission-handoff process resulted in improved verbal handoff quality and that physicians felt it facilitated better patient safety and efficiency. Improvements may have been limited by inconsistent application of the SBAR-DR format. Future areas of study could include the institution-wide implementation of the SBAR-DR model to avoid the use of competing handoff strategies and efforts to better engage practicing physicians prior to implementation.


Bamboo mosaic virus (BaMV), a member of the genus Potexvirus, is the major threat to bamboo cultivation. Similar to most potexviruses, the transmission of BaMV by insect vectors has not been documented previously. However, field observations of BaMV disease incidences suggested that insect vectors might be involved. In this study, we aimed to investigate the possibility of insect-mediated transmission of BaMV among bamboo clumps, in order to provide further insights into the infection cycles of BaMV for the development of effective disease management measures. From the major insects collected from infected bamboo plantations, BaMV genomic RNAs were detected inside the bodies of two dipteran insects, Gastrozona fasciventris and Atherigona orientalis, but not in thrips . Artificial feeding assays using green fluorescent protein-tagged BaMV virions revealed that BaMV could enter the digestive systems and survive in the regurgitant and excretion of the dipterans. BaMV RNA could be retained in the dipterans for up to 4 weeks. Insect-mediated transmission assays indicated that both dipterans could transmit BaMV to bamboo seedlings through artificially created wounds with low infection efficiency (14 – 41%), suggesting that the dipterans may mediate the transmission in a mechanical-like manner. These results demonstrated that dipterans with sponge-like mouthparts may also serve as vectors for at least one potexvirus, BaMV, among bamboo plants. The finding suggested that dipteran insect control should be integrated into the disease management measures against BaMV infections. Bamboo mosaic virus (BaMV), a member of the genus Potexvirus in the family Flexiviridae  that has been reported to be transmitted by aphids in a persistent mode  and the pruned culms attract large amounts of insects, mostly of the order Diptera, within minutes. The dipterans stay probing and feeding on the cut surfaces till the surfaces dry out if not disturbed, and fly on to the newly generated wounds of the bamboo clumps, possibly far away from the original clump as the harvesting or pruning processes proceed while the insects are feeding. If the dipterans are actually involved in BaMV transmission among bamboos, the lack of measures against insect pests may constitute a loophole in the current integrated disease management of BaMV.It has been considered that most of the potexviruses are not transmitted by insect vectors , with onent mode , althougent mode  as the hent mode . Similarent mode . HoweverIn this study, we aimed to explore the possibility that insects may mediate the transmission of BaMV, a potexvirus, among bamboos. Several lines of evidence were provided in support of the notion that at least two dipteran insects may serve as vectors for transmission of BaMV among bamboos. The results revealed the potential threat of the dipteran insects with sponge-like mouthparts as vectors for at least one plant virus, and suggested that the control of these insects should be integrated into the current systems for disease management against BaMV infections.Gastrozona fasciventris were anesthetized with acetone vapor and placed into 1.5-ml Eppendorf tubes containing 20 μl of BaMV  individually. The tubes were then incubated on ice for 5 min to coat BaMV onto the surfaces of the insects and the tubes. Following the removal of BaMV solution by manual pipetting, RNase Away (200 μl) was added to each tube. The tubes were then incubated at room temperature with gentle shaking for 1, 2, or 3 min, followed by thorough washing with 1 ml of de-ionized H2O for three times (3 min each). Then the samples were subjected to RNA extraction and analyses as described below. It was thus determined that the bamboo tissue and insect samples should be treated with RNase Away for 3 min to remove the contaminating nucleic acids on the surfaces before extraction of total nucleic acids (see Results). Total nucleic acids were extracted from bamboo or insect tissues according to the methods described previously  treatment was included as the first step of sample preparation. To determine the efficiency of RNase Away in removing the contaminating nucleic acids, BaMV-free eviously , with micytochrome oxidase I (COI), 28S rDNA and, mitochondrial 16S rDNA gene segments were amplified by PCR using gene-specific primer pairs (Table 1), cloned, and sequenced following standard protocols described in the Barcode of Life Database (Table 1). The PCR mixture (50 μl) contained 5 μl of 10× rTaq buffer, 5 μl of dNTP mixture (2.5 mM each), 0.25 μM of specific primer pair , 1.0 unit of rTaq DNA polymerase  and 1–2 μl of the aforementioned total nucleic acids or cDNA products as the template. PCR was performed as follows: reaction mixtures were heated to 94°C for 4 min for initial denaturation, followed by 35 cycles of denaturing at 94°C for 40 s, annealing at 47°C for 45 s, and extension at 72°C for 45 s, with a final extension at 72°C for 20 min. The PCR products were then analyzed by electrophoresis through a 1% agarose gel, stained with ethidium bromide (EtBr), and examined by UV illumination.To identify the dipteran insects collected from bamboo plantations, the chromosomal Database . For theTable 1), and inserted to the 5′-end of truncated CP ORF on pBS-d35CP following restriction digestion with BamHI and PspOMI. The GFP coding region (Table 1), digested with BamHI and then inserted to the 5′-terminus of the FMDV 2A coding region to give pCB-GFP2a-CP. Due to the presence of FMDV 2A cotranslational dissociation peptide, the inoculation of pCB-GFP2a-CP in plants would generate both GFP-fused and unfused BaMV CP to assemble into GFP-tagged BaMV virions, together with free GFP-2a fusion proteins. The incorporation of FMDV 2A peptide would increase the assembly efficiency and stability of the GFP-tagged BaMV virions, compared to the construct without FMDV 2A peptide . Leaves of Chenopodium quinoa mechanically inoculated with purified plasmid pCB-GFP2a-CP DNA  or wild type BaMV virions  were harvested at 10 dpi. The leaves were ground in de-ionized water (1:10 w/v) to prepare the inoculum used for mass-inoculation of C. quinoa plants (50–100 plants/batch). At 10 dpi, the leaves were harvested, and the GFP-tagged or wild type BaMV virions were subsequently purified from the leaves as described by To track the ingestion of BaMV virions, a mutant BaMV construct that generates GFP-tagged BaMV virions in plants, designated pCB-GFP2a-CP, was constructed based on a mutant infectious clone of BaMV, pBS-d35CP . The codg region  was amplBambusa oldhamii) seedlings originating from meristem tip-cultured bamboos . Two major insect species were identified to be Gastrozona fasciventris  and Atherigona orientalis , based on the morphology, wing markings  located in the major bamboo production areas in southern , central , and northern Taiwan . The BaMV disease incidences in these plantations ranged from 40 to 70%. RT-PCR was used to detect the presence of BaMV in the main insect species collected. RNase Away was used to avoid the contamination of BaMV on the outer surfaces. To determine the treatment conditions, a pre-test was performed using BaMV-coated G. fasciventris. As shown in Figure 2A, the treatment of RNase Away for 1 min was enough to remove the contamination of BaMV. For the following experiments, the insect samples were treated with RNase-Away for 3 min to ensure the removal of the possible contaminations of RNases and RNAs, and thoroughly rinsed with RNase-free de-ionized water before being subjected to total nucleic acid extraction. RT was performed using oligo-dT as the sole primer. The cDNA products were then subjected to PCR with primer pair specific to BaMV 3′-terminus (B-5981R and B-6366). The results revealed that BaMV could be detected in the dipteran insect samples, A. orientalis and G. fasciventris, but not in the thrips  collected from the backside of the leaves of BaMV-infected bamboos . The results of the BaMV detection in A. orientalis and G. fasciventris collected from the five bamboo plantations were summarized in Table 2. BaMV detection was not performed on T. vittigera, S. appendiculatus, and D. melanogaster, since these minor insects were not always trapped in all bamboo plantations surveyed, and thus not included in further analyses.The harvesting, pruning, and other regular maintenance of bamboo crops create cut surfaces or wounds that attract many insects within minutes, mostly in the order markings , and nucsegments . Other mG. fasciventris and A. orientalis on BaMV-free bamboo seedlings in 200-mesh insect domes. The insects and the bamboo seedlings were indexed bi-weekly to ensure the BaMV-free status (data not shown). These BaMV-free dipteran insects were used in the following assays.To further explore the possibility that the dipteran insects may mediate the transmission of BaMV, we established BaMV-free colonies of C. quinoa leaves inoculated with pCB-GFP2a-CP , which express GFP-fused BaMV CP. Examination of the purified GFP-tagged BaMV virions under UV illumination confirmed the presence of GFP-tags on the purified virions , as compared to the wild type BaMV virions . The dipterans were fed with liquid medium containing 0.1 mg/ml of purified GFP-tagged or wild type BaMV virions  in a inversed 50-ml Falcon conical tube for 24 h. The presence of GFP signals (likely representing the GFP-tagged BaMV virions) in dipterans were examined by using a LAS-4000 Chemiluminescence and Fluorescence Imaging System . The result revealed that green fluorescence was clearly visible in the abdomen portion of G. fasciventris fed with GFP-tagged BaMV virions , while those fed with wild type BaMV exhibited only background fluorescence. However, the green fluorescence might also be contributed by the unassembled GFP-tagged BaMV CP or GFP-2a proteins in the virion preparations, thus the presence of BaMV RNAs in the dipterans were further assayed. Following surface decontamination by RNase Away and RNA extraction, the presence of BaMV genomic RNA in A. orientalis and G. fasciventris fed with BaMV-containing medium was confirmed by RT-PCR . The observations suggested that BaMV may actually enter the digestive systems of dipterans, instead of just temporarily associated with the mouthparts of the insects as in the cases for non-persistent type transmission  with gentle shaking (60 rpm) for 3 min to elute the virus particles possibly adhered to the external surfaces. The eluate was then collected and subjected to total nucleic acid extraction as describe above. The washed insect bodies were centrifuged twice at 1000 × g for 10 s to remove excess wash buffer, and also subjected to total nucleic acid extraction. Total nucleic acids from insects fed on healthy plants were used as the templates for the negative controls. The presence of BaMV genomic RNAs was assayed by RT-PCR using primer pair B-5981R and B-6366. The result revealed that BaMV RNAs could only be detected in the insect bodies, not in the eluates of the external portions of A. orientalis and G. fasciventris . The above observations suggested that BaMV virions are not simply adhering to the surfaces of A. orientalis and G. fasciventris for dispersion by the mechanical contacts between insects and plants. To test the ability of the dipterans to retain BaMV RNAs, the dipterans were fed with BaMV-containing medium for 24 h, followed by feeding with virus-free medium for up to 6 weeks. Total RNAs were extracted from samples collected at 2-week intervals and subjected to RT-PCR analysis for the presence of BaMV RNA. The results showed that BaMV RNAs could be retained by G. fasciventris for up to 4 weeks . On the other hand, the BaMV RNA appeared to be degraded after 4 weeks in A. orientalis, but barely detectable amount of RT-PCR products was still observed at least in one of the samples .To verify the localization of BaMV on the dipterans, A. orientalis and G. fasciventris adults were starved for 4 h, and then fed with media containing GFP-tagged BaMV in 50-ml Falcon conical tubes for 24 h. The regurgitants and excretions of the flies on the wall of the tubes were collected by dissolving in nucleic acid extraction buffer (2 μl) under a dissecting microscope, and subjected to RNA extraction and RT-PCR analysis as described above. Although it has been shown that BaMV RNA could not be detected on the external surfaces of these insects , there is still the possibility that the walls of the tubes could be contaminated by GFP-tagged BaMV from the evaporation of the medium or the streaking of the insects. To test the possibility of contamination on the walls, aliquots of 2-μl nucleic acid extraction buffer were used to elute any possible contaminants present on the areas surrounding the regurgitant and excretion droplets. The eluates  were then subjected to nucleic acid extraction and RT-PCR analysis concurrently with the regurgitant and excretion samples, serving as negative controls. To overcome the problem of relative inefficient amplification of full-length GFP-tagged BaMV RNAs (∼7.0 kb) directly by RT-PCR using primers 1R and oligo dT, an alternative approach was adopted by using different primer pairs  to detect different portions of the GFP-tagged BaMV RNA throughout the entire length, using the first-strand cDNA synthesized using the oligo-dT primer as the only template. Since the first strand cDNA is synthesized using oligo-dT primer starting from the 3′-poly(A) tail of BaMV genome, the amplification of the different fragments, especially the one corresponding to the very 5′ terminus (amplified by primers 1R and 331), indicated that the intact BaMV genomic RNA was present in the sample. The results revealed that intact BaMV RNAs could survive the digestive enzymes in the regurgitants and pass through the digestive system to reach the excretion portion of both A. orientalis and G. fasciventris . In contrast, no BaMV RNA was detected in the eluates from the wall areas surrounding the regurgitant and excretion droplets . The above observations raised the possibility that BaMV might be transmitted by the dipterans through the regurgitants or excretions while probing and feeding on bamboos.For a virus to be transmitted by an insect vector following ingestion into the abdomen, the virus must survive and pass through the digestive system of the insect. When dipterans feed, they constantly regurgitate crop fluids with digestive enzymes to help dissolve the substrate . The excA. orientalis or G. fasciventris fed with liquid media containing wild type BaMV were used to inoculate the bamboo seedlings. The dipterans were allowed to feed freely for 24 h, and then removed manually. The newly emerged leaves from the new shoots of the inoculated bamboo seedlings were assayed for the presence of BaMV RNA by RT-PCR at day 60 post inoculation. The results of dipteran insect-mediated BaMV transmission assays were summarized in Table 3. The representative result  demonstrated that G. fasciventris may actually mediate the transmission of BaMV to bamboo seedlings. To test the possibility of BaMV contamination in the tested plants before the inoculation, we have traced back and examined the un-inoculated siblings from the same mother-stock of the seedlings which tested positive for BaMV after the completion of Experiment 4 (Table 3) by northern  and western blot  analyses using 32P.-labeled probe specific to the 3′ untranslated region of BaMV RNA  which tested positive for BaMV in Experiment 4 (Table 3) were also assayed concurrently for the products of BaMV infections in the newly emerged leaves. As shown in Figures 6B,C, the un-inoculated siblings from the same mother stock (lanes 1–4) remained BaMV-free, while the subgenomic RNAs and TGBp1 protein, which are produced only after the successful infection of BaMV, could be detected in those tested positive (lanes 5–7) in the insect-mediated inoculation assays .To test the abilities of the dipterans in transmitting BaMV among bamboo plants, the following experiments were conducted. To simulate the possible insect-mediated transmission during harvesting process, the newly emerged bamboo shoots were cross-sectioned at the crowns to create wounds for insect-mediated virus transmission assays. Since the fusion of the GFP at the N-terminus of BaMV CP interfered with the infectivity in bamboos, the dipterans were fed with liquid media containing purified wild type BaMV virions (0.1 mg/ml) for 24 h, and then subjected to inoculation assays by co-incubation with the bamboo seedlings with cross-sectioned shoots in 200-mesh insect domes. Within each dome, four to seven BaMV RNA  and antiBaMV RNA , respectBased on the field observations, we explored the possibility of insect-mediated transmission of BaMV among bamboos in this study. Several lines of evidence were provided to support the notion that dipterans may serve as the vector for BaMV, including the association of BaMV with the internal portion of the dipterans, the survival of BaMV RNA through the digestive systems, and the inoculation assays on bamboo seedlings. Our study revealed a novel finding with regards to the role of dipterans as vectors for a plant virus, and the insect-mediated transmission of a potexvirus.Tobacco mosaic virus or Potato virus X. However, to our knowledge, no dipterans with sponge-like mouthparts have been reported to transmit plant viruses prior to this study.For the role of dipterans as vectors for a plant virus, the flies have been known to transmit many animal pathogens, including viruses see . This moPotato virus X and White clover mosaic virus has been reported to be transmitted by grasshoppers and aphids, respectively , remain associated with the dipterans for up to 4 weeks, and survive in the regurgitants and excretions , then sucking the fluids without laying eggs. During the probing and feeding process, the dipterans with sponge-like mouthparts would regurgitate crop fluid and deposit excretions around the feeding sites (Table 3) and the involvement of artificially created wounds in the assays suggested that the dipteran insects may transmit BaMV in a mechanical-like manner, not directly feeding BaMV into the plants. Further studies are required to fully analyze the relationship between BaMV and the dipterans and the mode of transmission involved.Female phytophagous flies are known to feed by piercing the surface of plants by the ovipositors (as seen on the posterior portion of ng sites . The digAs mentioned above, no known insect vectors have been reported for BaMV , thus thThe finding in this study that dipterans may mediate the transmission of BaMV highlights the importance of the integration of dipteran insect control and prevention measures into the current disease management system against BaMV. Since the dipterans tested in this study exhibited much longer virus acquisition feeding time, virus retention time, and transmission feeding time, compared to those of the non-persistent mode of transmission, the transmission mode of BaMV by the dipterans may be categorized as at least “semi-persistent,” if not “persistent.” Thus, the dipterans may be controlled by using suitable pesticides, without the concern of causing increased dispersals of the viral diseases as seen for “non-persistent” mode of transmission.To our knowledge, this is the first report describing the transmission of a plant virus by dipterans with sponge-like mouthparts, These results expanded the types of insects as vectors for plant viruses, and suggested that dipteran insect control should be concerned and integrated into the disease management measures against viruses, such as BaMV, that are structurally stable enough to survive through the digestive systems of these insects.Study conception and design: Y-HH, N-SL, and C-CH. Acquisition of data: K-CC, L-TC, Y-WH, Y-CL, C-WL, J-TL, and C-CH. Analysis and interpretation of data: Y-HH, N-SL, Y-WH, and C-CH. Drafting and critical revision of the manuscript: Y-HH, N-SL, Y-WH, and C-CH.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.


Treatment success rates for multidrug-resistant tuberculosis (MDR-TB) in South Africa remain close to 50%. Lack of access to timely, decentralized care is a contributing factor. We evaluated MDR-TB treatment outcomes from a clinical cohort with task-sharing between a clinical nurse practitioner (CNP) and a medical officer (MO).We completed a retrospective evaluation of outcomes from a prospective, programmatically-based MDR-TB cohort who were enrolled and received care between 2012 and 2015 at a peri-urban hospital in KwaZulu-Natal, South Africa. Treatment was provided by either by a CNP or MO.The cohort included 197 participants with a median age of 33 years, 51% female, and 74% co-infected with HIV. The CNP initiated 123 participants on treatment. Overall MDR-TB treatment success rate in this cohort was 57.9%, significantly higher than the South African national average of 45% in 2012 (p<0·0001) and similar to the provincal average of 60% (p = NS). There were no significant differences by provider type: treatment success was 61% for patients initiated by the CNP and 52.7% for those initiated by the MO.Clinics that adopted a task sharing approach for MDR-TB demonstrated greater treatment success rates than the national average. Task-sharing between the CNP and MO did not adversely impact treatment outcome with similar success rates noted. Task-sharing is a feasible option for South Africa to support decentralization without compromising patient outcomes. Models that allow sharing of responsibility for MDR-TB may optimize the use of human resources and improve access to care. Multidrug-resistant tuberculosis (MDR-TB) is a global epidemic, threatening progress towards the World Health Organization’s (WHO) Global Plan to Stop TB . AccordiMany countries have opted for centralized, hospital-based care in regional referral centers to manage drug-resistant TB. This strategy falls short in high-prevalence areas where many participants are concentrated in rural areas with limited hospital bed capacity . In 2011One potential solution to this human resource challenge is task-sharing, a hybrid and more cooperative form of task shifting, which is defined as the movement of medical officer-driven clinical tasks to nurses with appropriate training . Task-shA single, peri-urban district hospital with a stand alone MDR-TB treatment unit in KwaZulu-Natal was chosen. The MDR-TB complex, included a 40 bed MDR-TB inpatient unit equally divided between male and female wards. The outpatient clinic for MDR-TB is situated adjacent to the inpatient ward and the hospital’s outpatient HIV clinic. The majority of patients at the site are admitted to the hospital ward for treatment initiation, however this varied based on bed capacity. The site follows the South African national treatment guidelines for clinical management. The catchment population for the hospital is noted to be 226,008 with six feeder primary care clinics. It was the only MDR-TB treatment initiation site in the district at the time of the study .Mycobacterium growth indicator tubes (MGIT) followed by drug sensitivity testing (DST) on positive samples. All samples were processed through the National Health Laboratory System. Patients diagnosed with MDR-TB received the clinical standard of care for MDR-TB treatment in KwaZulu-Natal, South Africa, which for this area of the country includes a six-drug regimen [Diagnosis of drug-resistant tuberculosis was made using standard culture-based approaches with  regimen . TreatmeParticipants were included in the programmatically-based observational cohort if they started MDR-TB treatment between January 3, 2012 and December 31, 2012. Upon cohort completion, all participant charts were reviewed until the point of outcome, which included all treatment completion and cure patients up to 24 months of follow-up. The final completion of treatment on all participants was January 2015. In May 2015, the treatment outcomes for all participants, received a second review and verification by the study team. The study was approved by The Johns Hopkins University Institutional Review Board in Baltimore, Maryland, USA and the Medical Research Council Ethics Committee in Pretoria, South Africa. The study was reviewed and approved by the Centers for Disease Control and Prevention under the protocol entitled, “CO-INFECT Project: Nurse Case Management”.2 automatically received care from the medical officer due to severity of illness on presentation, which was a programmatically-based decision. All participants were given the option to be followed by the MO during the standard hospital consent process for treatment if they did not wish to have their care completed by the CNP.All patients presenting for MDR-TB care with culture-based evidence of isoniazid and rifampicin resistance were eligible to receive care from either provider and assignment was based on availability of the next provider. Study investigators had no role in treatment assignment. Participants, however, who presented with a baseline body mass index (BMI) less than 18 kg/mClinical duties for both providers included all required care during the treatment period, including inpatient services. Face to face and telephonic clinical consultation with the medical officer was available and completed as needed from the CNP throughout the course of treatment. In general, however, each clinician managed their assigned caseload unless out of the office, at which time the other clinician provided back-up clinical coverage for the absent member’s caseload. As part of current standardized care in South Africa, all patients with MDR-TB sign a one-page indemnity form for treatment that details the treatment regimen, provider options and basic expectations associated with the treatment course.Treatment outcomes based on the South African NDoH guidelines are determined by sputum culture results and include treatment success (cure/completion of therapy), death (for any cause), failure (worsening drug resistance necessitating change in treatment), and default, which is now known as loss to follow-up (interruption of treatment for two consecutive months) . ProcessThe CNP’s prerequisite training included an advanced diploma in primary healthcare management  and training in nurse-initiation and management of anti-retroviral therapy (NIMART). The MO’s prerequisite training included a 5-year Bachelor’s of Medicine (MBBCh) with an additional year of community service and training in HIV clinical management through an Advanced Post Graduate Diploma. The MO had also completed a one-week skills building short-course developed by The Johns Hopkins University School of Nursing in the diagnosis, treatment, and management of MDR-TB. Both the MO and CNP had extensive prior experience in the study hospital site. After hire, the CNP participated in the same one-week short course as the MO followed by an intensive one-week physical exam and health assessment skills update in the MDR-TB unit. Study team members with substantial MDR-TB clinical knowledge use a standardized competency-based tool, developed by the study team, to validate the CNPs baseline clinical skills through direct observation. After this evaluation the CNP received a one-month period of clinical mentoring by MOs at two regional drug-resistant TB centers in the province. Ancillary training and clinical mentoring were provided to the CNP during this period of supervised clinical mentorship in the following MDR-TB activities: chest x-ray evaluation and interpretation, laboratory monitoring, adverse treatment effects and their management as well as audiometry monitoring and interpretation.We computed descriptive statistics of the sample overall, and by provider type (CNP vs. MO). Medians  were calculated for skewed continuous variables and frequencies (%) were calculated for categorical variables. A Wilcoxon rank-sum test was used to compare the two groups (CNP vs. MO) for continuous patient characteristics and outcome variables and a Chi-square test was used to compare proportion of treatment outcome type between provider types. A one-sample z-test was used to compare the success rate in this cohort with the national programmatic outcomes data from South Africa. The treatment outcome was analyzed as proportion of outcome and also as time to an event. A multivariate Cox proportional hazards model was used to examine the association between provider type and time to a treatment outcome to evaluate type-specific hazards controlling for age, sex and baseline HIV status . SignifiTwo-hundred twelve participants presented with MDR-TB for treatment at this site. Fifteen participants (7%) transferred their care to another facility making them ineligible for the final cohort analysis. One hundred ninety-seven participants, 101 (51.3%) female, with a median age of 33 years (IQR = 26–40) and a median body mass index (BMI) of 19.7 (IQR = 16.4–22.7) were enrolled. HIV status at enrollment was known for all but one patient with 145 (74%) co-infected with HIV  and of those, 108 (74.5%) were on ART at enrollment. The majority of participants  were admitted to the hospital for treatment initiation. BMI was a significantly different (p = 0.049) variable at baseline between the two providers  which maTreatment success for the cohort was 57.9% and significantly higher than the 45% national norm  for the 2012 South African cohort amongst 8,435 patients and similar to the 60% success rate for KwaZulu-Natal province amongst 3,190 patients (p = NS). Eleven (5.6%) participants experienced treatment failure, 33 (16.8%) died, and 39 (19.8%) were lost to follow-up . CompariIn a prospective, programmatically-based, observational cohort, in a peri-urban site with over 70% HIV co-infection, a task-sharing approach for MDR-TB demonstrated greater treatment success rates (57.9%) than the national average 45%).Task-sharing between the providers did not adversely impact treatment outcome. Process indicators and clinical outcomes were similar between providers, with the one exception of length of stay, which was shorter in the MO-led group. These findings demonstrate that nurse-led treatment did not compromise patient care. These findings support the potential for task-sharing for MDR-TB treatment initiation and management between medical officers and clinical nurse practitioners to work in some settings. Participants with HIV and increasing age had an increased hazard for death, which was independent of provider assignment and consistent with previously published literature –4.5%.Task-sThese findings add further support to previous studies conducted in under-resourced African settings that utilize nurse-led models of care to manage HIV infection –17. DeceWhile this evaluation of a CNP-MO MDR-TB program is the first of its kind and demonstrates programmatic outcomes exceeding those at the country level, limitations exist. The study was limited to retrospective abstraction of clinical records and documentation describing the clinical care for each patient. While this may have limited impact on the final treatment outcome determination, it limits the standardization of the type, frequency and preciseness one might anticipate in a more controlled research evaluation. To avoid missing data–mainly from lost or damaged medical files—our research team visited the site on a quarterly basis to retrospectively abstract data from the medical record. We also know that the treating clinical team was limited, and consisted of only one MO and one CNP initiating and managing MDR-TB treatment at the clinic, a circumstance not unusual in the South African context. This resulted, however, in the MO and CNP supporting one another during periods of illness or extended leave of absence. Although this limited the granular nature of our comparison, we felt that this improved generalizability and was ultimately a strength of the task-sharing model in a real-world setting, as the sharing of patient responsibility benefited both clinicians. The cross coverage needs that arose from the real world human resource challenge that exist resulted in approximately four months where the CNP was the sole clinician and another two months where the MO was the sole clinician. In both circumstances, participants received care outside of their original patient assignment. It is important to note that without such a model the MDR-TB program would have been without a trained MDR-TB care provider for approximately four months in an MO-only model. In the year prior to the programmatic implementation of this approach, this site experienced a period of more than six months without a trained clinician responsible for managing MDR-TB participants. It is important to note that no increase in death, default nor failure was noted during the time the CNP provided sole coverage. While the CNP can take on much of the patient burden thereby freeing the MO for care of more complicated MDR-TB cases, this circumstance channels sicker patients into the MO’s care and may bias outcome comparisons. In this cohort, the MO did manage participants who were significantly underweight (BMI < 18) on presentation to care, but this did not lead to differences in treatment outcome. It does likely account for the slightly lower success rates and lower inpatient days for the MO. Another possible explanation for this finding is that the MO had greater clinical experience and therefore felt more comfortable releasing the patient from the hospital earlier. Finally, as an observational study, this descriptive analysis does not offer the necessary evidence to support large scale implementation; however these data are promising and suggest the need for additional randomized evaluations.Task-sharing is a feasible option for the peri-urban areas in South Africa to support decentralization efforts without compromising patient outcomes. Further evaluation through randomized controlled trials is required to more fully determine the impact of nurse-led models of MDR-TB care. As access to care remains a priority, the South African NDoH is presently decentralizing care to community health centers using the nurse-led model of care that has been presented in this study –29.


Somatic mutations in cancer genomes often show allelic imbalance (AI) of mutation abundance between the genome and transcriptome, but there is not yet a systematic understanding of AI. In this study, we performed large-scale DNA and RNA AI analyses of >100,000 somatic mutations in >2,000 cancer specimens across five tumor types using the exome and transcriptome sequencing data of the Cancer Genome Atlas consortium. First, AI analysis of nonsense mutations and frameshift indels revealed that nonsense-mediated decay is typical in cancer genomes, and we identified the relationship between the extent of AI and the location of mutations in addition to the well-recognized 50-nt rules. Second, the AI with splice site mutations may reflect the extent of intron retention and is frequently observed in known tumor suppressor genes. For missense mutations, we observed that mutations frequently subject to AI are enriched to genes related to cancer, especially those of apoptosis and the extracellular matrix, and C:G > A:T transversions. Our results suggest that mutations in known cancer-related genes and their transcripts are subjected to different levels of transcriptional or posttranscriptional regulation compared to wildtype alleles and may add an additional regulatory layer to the functions of cancer-relevant genes. Advances in high-throughput genome sequencing technology have facilitated the sequencing of hundreds of thousands of cancer genomes, resulting in the identification of many cancer-related somatic mutations4. SNVs and indels comprise a major fraction of somatic alterations in cancer genomes, especially those profiled by recent sequencing efforts. Their functional consequences have been inferred based on the amino acid alterations resulting from nucleotide-level changes. For example, truncating mutations including nonsense SNVs and frameshift indels as well as those occurring at splice sites are considered loss-of-function events, while synonymous or silent SNVs are considered functionally neutral. In the case of nonsynonymous or missense SNVs, a number of hotspot mutations with clinical relevance have been identified 6. A number of methods have also been proposed to infer the potential functional consequences of missense mutations 9. However, it is a daunting task to identify the functional consequences of the missense mutations that comprise a major fraction of somatic alterations in cancer genomes.Cancer is a genomic disorder associated with various types of somatic alterations from small-scale single-nucleotide variations or substitutions (SNV) and short insertions/deletions (indels) to chromosome-scale copy number alterations and rearrangements11. The comparison of allelic frequencies of somatic mutations at DNA and RNA levels as measured from genome and transcriptome sequencing, respectively, can be used to assess the potential cis-impact of somatic mutations at the transcript level. The over- or under-representation of RNA transcripts harboring mutant alleles over wildtype alleles may be observed against the DNA-level mutant vs. wildtype allelic ratio, and this may indicate the presence of genetic mechanisms that are in favor of or against the mutant transcripts at the transcription or post-transcription level. It is well known that RNA transcripts with premature termination codons (PTC) are subject to nonsense-mediated decay (NMD), in which the transcripts with PTC will be degraded by the RNA surveillance mechanism12. The preference of NMD can be determined by the PTC position. If the PTC is located within 50 nt from the rightmost exon-exon junction or located at the last exon, the transcript may escape from NMD (50-nt rule)13. NMD associated with germline variants has been recently demonstrated in large-scale paired transcriptome-genome sequencing data14. The roles of NMD in carcinogenesis have been proposed previously15, and its prevalence in cancer genomes has been recently investigated, focusing on ~3,000 nonsense mutations16. In addition, it has been proposed that non-mutated variants may be subject to NMD, suggesting that NMD is more prevalent than previously expected especially in cancer19. In addition, mutant-biased transcriptional up-regulation was observed for some missense SNVs such as those on EGFR and KRAS21. This observation suggests that some cancer-related somatic mutations are subjected to different levels of transcription or post-transcriptional regulation; however, the systematic understanding of such allelic imbalance (AI) between the genome and transcriptome of somatic mutations in cancer genomes is still lacking.Although the impact of the somatic mutation on the gene expression has been poorly understood, it is supposed that the somatic mutation may influence on the stability of mutant transcriptsvs. wildtype allelic imbalance as measured between DNA and RNA levels, where the DNA- and RNA-level mutation abundances are measured from whole-exome and transcriptome sequencing data). The main objective of our study was to investigate the impact of major types of somatic mutations in terms of AI reflecting their potential transcriptional or post-transcriptional regulatory mechanisms. For the study of AI, we collected sequencing data from the large-scale cancer genome database of the Cancer Genome Atlas (TCGA) consortium. Five major human tumor types  of >2,000 patients were used in this study. We collected a large number of somatic mutations and performed AI analyses with respect to the mutation categories of missense, silent, nonsense, and splice site SNVs along with frameshift and inframe indels. Our systematic analysis revealed that the somatically mutated genes often showed AI of transcription, and the preference for allelic expression was diverse across the mutation types.In this study, we performed a large-scale AI analysis of the somatic mutations in cancer genomes . We categorized the somatic mutations into four SNV classes  and two indel classes (frameshift and inframe), respectively. The mutant and wildtype allele counts of individual somatic mutations at DNA and RNA levels were also obtained from TCGA tumor BAM files as whole-exome sequencing (WES) and transcriptome sequencing (RNA-seq), respectively. We further selected the mutations supported by no less than 10 sequencing reads both in WES and RNA-seq datasets to obtain initial candidates of AI analyses. Finally, we selected 103,661 mutations  observed in 13,687 genes and 2,112 tumor specimens. The preprocessing procedure is illustrated in Supplementary Figure For AI analyses, we first collected a total of 368,921 somatic mutations  from 2,243 tumor specimens across five tumor types  from the TCGA portal  of the DNA and RNA from the mutant and wildtype allele counts of WES and RNA-seq, respectively. The distributions of RNA-VAF and DNA-VAF of all the mutations across the five tumor types are shown in Fig. AFs Fig. . The bia13, we further categorized the nonsense SNVs and frameshift indels into NMD-sensitive mutations  and NMD-insensitive mutations. The extent of AI was more apparent for NMD-sensitive mutations than NMD-insensitive mutations both for the nonsense SNVs and frameshift indels, suggesting that the 50-nt rule for NMD-sensitivity is true for cancer genomes . No significant difference was observed between the DNA-VAF and RNA-VAF for NMD-insensitive nonsense SNVs, frameshift indels, or other variants including missense SNVs, silent SNVs, and inframe indels.The preference of negative AFD for nonsense SNVs and frameshift indels is consistent with a known biological phenomenon of NMD where the transcripts containing those truncating mutations are relatively deficit compared to those with wildtype alleles. According to the 50-nt rulemes Fig. . The RNAet al.23. As expected, the proportion of sig-neg-AFD mutant transcripts representing NMD events was significantly higher for the nonsense mutations than for other somatic mutation classes . The scatterplots of RNA-VAF and DNA-VAF for the NMD-sensitive and -insensitive nonsense SNVs and frameshift indels also show the relative enrichment of sig-neg-AFD mutations in NMD-sensitive mutations compared to NMD-insensitive mutations  were observed in TP53. Moreover, for SNVs, the significant enrichment of sig-neg-AFD mutations were observed in the NMD-sensitive nonsense SNVs compared to other classes including the NMD-insensitive nonsense SNVs across the five tumor types . Similarly, the abundance of sig-neg-AFD was significantly higher for NMD-sensitive frameshift indels compared to inframe indels and frameshift indels with no PTC or NMD-insensitive PTC across the tumor types examined . To further investigate the 50-nt rules, we compared the proportion of sig-neg-AFD in four entities according to the occurrence of PTC  . In addition, the proportion of sig-neg-AFD measured in 10 bp-bins of the distant between PTC and the last exon junction further supports that the 50 nt rule is an important factor in NMD efficiency  as previously described by Rashid rs) Fig. . The proncy Fig. .P value 1.66 × 10−5; Fisher’s exact test). The relatively small fraction of 12.10% (19 nonsense mutation) of nonsense SNVs in the first exons among sig-neg-AFD events suggests that the PTC in the first exons is less vulnerable to NMD, which is consistent with the idea that the transcript harboring PTC close to the start codon may be insensitive to NMD26. For nonsense mutations in the first exon, the distance from the start codon was investigated, and we found that sig-neg-AFD events were significantly distant from the start codon compared to others . No significant difference in the mutation spectra was observed with respect to the mutation abundance of sig-pos-AFD-vs.-others . Among 223 sig-pos-AFD splice site mutations, 29 and 4 mutations were observed in 8 TSGs and 4 oncogenes, respectively. Sixteen mutations were found on 4 genes both annotated as oncogenes and TSGs including 13 mutations in TP53 gene. Figure TP53, CDKN2A, STK11, and ARID1A frequently harbored sig-pos-AFD at splice site mutations. Although HNRNPK was not included in the cancer gene census as a TSG, it may be a haploinsufficient tumor suppressor in hematologic malignancies30. This finding suggests that the major targets of sig-pos-AFD at splice site mutations are putative TSGs leading to the aberrant splicing  of them as one of potential inactivating mechanisms of TSGs29.Aberrant splicing may play a pathogenic role in a variety of human diseases including cancersKRAS show elevated transcript level compared to wildtype21. Across five tumor types, 11.1–20.4% and 6.4–11.4% of missense mutations were classified as sig-pos-AFD or sig-neg-AFD, respectively, suggesting that a substantial number of missense mutations are associated with AI , showing an overrepresentation of C:G > A:T in sig-neg-AFD mutations (transversions) compared to other mutations. Consistently, when we compared the number of transitions and transversions for the missense mutations, the frequency of transitions at sig-pos-AFD was also significantly higher , excluding KIRC, as also previously observed in another tumor31. Among the genes, GATA3 was specifically founded in BRCA (P = 0.0028). GATA3 is the most abundant transcription factor in luminal epithelial cells, and its potential role in breast epithelial carcinogenesis has been recently highlighted32. As a histone modifier and mitochondria-related autophagy inhibitor, the therapeutic potentials of KDM4A and LRPPRC have been noted34. In addition to TP53, two TSGs, CASP8 and KEAP1, frequently showed sig-pos-AFD  in HNSC. CASP8 initiates programmed cell death as a well known TSG along with KEAP1, which is known to regulate oncogenic factor NRF235. SMARCA4 (P = 2.78 × 10−3) and KEAP1 (P = 4.93 × 10−5) were found to be significantly associated with sig-pos-AFD in LUAD. For STAD, a known TSG, CDH1, was also found to harbor significantly frequent sig-pos-AFD (P = 1.28 × 10−5), and it has been shown to have roles in multiple cancers including gastric cancer37. These results suggest that the preferential expression of mutated alleles is frequent for known or potential tumor suppressor genes. Although we filtered out the mutations on copy number gained or lost regions, it is still possible that low frequent or subclonal copy number deletions that are frequently associated with TSGs may explain the frequent AI of TSGs. Further investigation is necessary to determine whether there exists a transcriptional or post-transcriptional mechanism in favor of transcripts harboring mutations of TSGs. Although the sig-pos-AFD enriched genes are largely tumor suppressive, HRAS and ERBB2, which are as well-known canonical oncogenes, are observed to be significantly enriched with sig-pos-AFD missense mutations , we note that a substantial number of the genes with frequent sig-pos-AFD AI events are cancer-related genes. For example, Fig. 38. GSEA results showed that the genes with AI, especially sig-pos-AFD, were mostly enriched in the functional genesets representing apoptosis or cell differentiation cis-transcriptional regulatory effects of cancer mutations. A statistical model was employed to evaluate the imbalance of the number of reads with mutant and normal alleles in the exome (DNA) and transcriptome (RNA) sequencing data from the same patient. A major advantage of this approach is that the two alleles are measured from identical samples, and it can minimize the potential impact of technical issues such as tumor purity and copy numbers. Without assuming the copy number states of somatic mutations, we directly compared the proportion of variants allele measured by the sequence reads between DNA-based and RNA-based sequencing datasets.The plethora of cancer genome and transcriptome sequencing data in the public domain has opened opportunities to perform large-scale AI studies analyzing the mutant allele abundance between DNA and RNA. In this study, we assessed the extent of AI of cancer-related somatic mutations across multiple types of cancers to gain additional insights into the potential 13. Our study demonstrates that the 50-nt rules of the EJC model are also true for cancer genomes, and NMD may serve as an important mechanism to inactivate genes harboring truncating mutations, especially TSGs. In addition, we observed that PTC present outside the penultimate exon  might be associated with NMD according to distance from the start codon or from the downstream exon junctions. These findings enrich our insights into the relationship between the location of PTC and NMD in addition to the well-recognized 50-nt rules. For splice site mutations, those at the first intronic nucleotide (+1G) are the most enriched with sig-pos-AFD events, suggesting that these splice site mutations are more likely to experience intron retention. Our results imply that the G nucleotide at the 5′-end of the intron may be critical in the splicing function, and the mutations of that position will convert the sites into weaker splice sites39. Along with the splice site mutations with AI that are enriched in TSGs, we also observed that allele-specific expression at loci with missense mutations are common in known cancer-related genes. Although the genetic mechanisms that are responsible for the AI in missense mutations should be further investigated, known cancer-related genes with mutations may be subject to different transcriptional or posttranscriptional regulation leading to AI.First, we demonstrate that AI analysis with respect to different types of somatic mutations may be used to evaluate previously recognized biological phenomena. For example, 50-nt rules and exon-junction complex (EJC) models are well-recognized for NMD where the EJC proteins bind at least 50 nt upstream of the last exon junctions and will remain bound during protein translation and trigger the degradation of transcripts with PTC41, those supported by no less than 10 DNA (WES) and 10 RNA (RNA-seq) reads were subsequently analyzed for AI. Since WES and RNA-seq may give uneven coverage across the loci largely due to the extent of DNA capture and the level of transcription, respectively, this may affect the confidence of our AI analysis that is based on a Bayesian statistical test. Thus, we performed additional experiments to investigate the impact of minimum sequencing depth cutoff differentially set for WES and RNA-seq. Supplementary Figure In this study, we set the minimum sequencing depth of mutations for AI analysis to be 10. Thus, among the mutations found in WES by conventional mutation calling algorithmcis-regulatory variation43, whole-genome scale AI analysis including noncoding regions will be required to elucidate the allele-specific expression beyond the coding regions. Therefore, further investigation combining other genomic and epigenomic factors including DNA methylation or three-dimensional DNA conformation will be helpful to understand the complete processes for AI and to shed light on clinical applications.Since we focused on somatic mutations in coding regions as covered by WES, the AI in promoter or other non-coding regions was not evaluated. As previous studies have proposed that a substantial number of genes may have allele-specific expression with 44, head and neck carcinoma (HNSC)45, kidney renal clear cell carcinoma (KIRC)46, lung adenocarcinoma (LUAD)47, and stomach adenocarcinoma (STAD)48 from the TCGA consortium for AI analyses. First, we obtained somatic mutations from MAF (mutation annotation format) files generated by TCGA consortium and curated by TCGA Analysis Working Groups . The data for BRCA, HNSC, LUAD, STAD was generated by Illumina Genome Analyzer IIx. But, for KIRC, the data was generated by Illumina Genome Analyzer IIx, Illumina HiSeq, and ABI SOLID systems. To obtain allele counts of individual somatic mutations at DNA and RNA levels, we downloaded TCGA tumor BAM files as WES and RNA-seq in dbGaP (http://www.ncbi.nlm.nih.gov/gap)49 with controlled access authorization. The sequencing data have been recently moved to Genomics Data Commons Data Portal . The platforms used to generate raw sequencing data were Illumina GenomeAnalyzerII or HiSeq. Mutant or wildtype allele count or the number of sequencing reads supporting the mutations was obtained using Samtools with mpileup option after removing the sequencing reads with low mapping quality with the option of mpileup -q 150. The mutations covered by no less than 10 WES and 10 RNA-seq reads were used for the subsequent AI analyses. The annotation of somatic mutations in MAF files were re-examined by ANNOVAR51. We discarded mutations with inconsistent annotations between the ANNOVAR- and original MAF-based annotations and also discarded mutations in genes harboring multiple mutations. The hypermutated cases  were also excluded from the AI analyses. The NMD-sensitive/-insensitive nonsense mutations were discriminated as those >50 bp and <50 bp with respect to the most 3′ exon-exon junction. The same discrimination was made for indels using SIFT-annotated PTC location52. The overall procedures and filtering schemes are illustrated in Supplementary Figure We selected the five tumor types of breast cancer (BRCA)vice versa for the mutations with negative AFD . The significance of the difference in DNA-VAF and RNA-VAF was estimated by a Bayesian hypothesis testing framework as previously described by Rashids et al.23. The threshold of significance for the allelic differential expression was determined as Bayes factor > 10, as previously recommended. Finally, we defined sig-pos-AFD and sig-neg-AFG mutations as those of pos-AFD and neg-AFD with Bayes factor > 10, respectively. To select the copy number neutral mutations, we obtained the copy number segmentation files of Affymetrix SNP6.0 filtered for the germline copy number variations. By intersecting the copy number segments and somatic mutations of the same patients, the mutations that belonged to segments with log ratios > −0.15 and <0.15 were considered copy number neutral mutations and used for the AI analyses. For investigation of factors related to NMD, mRNA half-life information was acquired from Liderboom’s study16.VAF represents the fraction of the number of reads with variants or mutant alleles in the total number of reads at the mutated sites. For individual mutations, DNA- and RNA-VAFs were obtained from WES and RNA-seq, respectively. The AFD is defined as the RNA-VAF minus DNA-VAF in the range of . We further defined the mutations with positive AFD  as those with the mutant alleles preferentially expressed compared to wildtype alleles, and P value obtained from a one-tailed Fisher’s exact test.The functional analysis of genes with sig-pos-AFD and sig-neg-AFD missense mutations was carried out using preranked version of GSEA. A joint set of MSigDB c2cp  and c5 (Gene Ontology) was used as the functional gene set for the analysis. The input data, which was a ranked gene list, was compiled using the negative log Supplementary FiguresSupplementary Tables


Ctenomys tuco-tucos. We characterized the LRV of the Anillaco Tuco-Tuco (Ctenomys sp.) using recordings from free-living individuals and described the behavioral context in which this vocalization was produced during laboratory staged encounters between individuals of both sexes. Long-range calls of Anillaco tuco-tucos are low-frequency, broad-band, loud, and long sounds composed by the repetition of two syllable types: series (formed by notes and soft-notes) and individual notes. All vocalizations were initiated with series, but not all had individual notes. Males were heavier than females and gave significantly lower-pitched vocalizations, but acoustic features were independent of body mass in males. The pronounced variation among individuals in the arrangement and number of syllables and the existence of three types of series , created a diverse collection of syntactic patterns in vocalizations that would provide the opportunity to encode multiple types of information. The existence of complex syntactic patterns and the description of soft-notes represent new aspects of the vocal communication of Ctenomys. Long-distance vocalizations by Anillaco Tuco-Tucos appear to be territorial signals used mostly in male-male interactions. First, emission of LRVs resulted in de-escalation or space-keeping in male-male and male-female encounters in laboratory experiments. Second, these vocalizations were produced most frequently (in the field and in the lab) by males in our study population. Third, males produced LRVs with greater frequency during male-male encounters compared to male-female encounters. Finally, males appear to have larger home ranges that were more spatially segregated than those of females, suggesting that males may have greater need for long-distance signals that advertise their presence. Due to their apparent rarity, the function and acoustic features of LRV in female tuco-tucos remain inadequately known.The underground environment poses particular communication challenges for subterranean rodents. Some loud and low-pitched acoustic signals that can travel long distances are appropriate for long-range underground communication and have been suggested to be territorial signals. Long-range vocalizations (LRVs) are important in long-distance communication in  Ctenomys spp.) are fossorial rodents with over 50 species found in the southern half of South America  based on recordings made from free-living males and females from a population in La Rioja province, Argentina, and; 2) describe the behavioral context in which this vocalization is produced during laboratory experiments designed to assess the communicative function of this signal. In addition to contributing to our knowledge of the nature of acoustic communication in Ctenomys, these analyses generate support for the hypothesis that long-range acoustic signals in solitary species of tuco-tucos function in territory defense.This paper aims to: 1) provide the first quantitative acoustic characterization of the LRV of the Anillaco Tuco-Tuco , La Rioja, Argentina. The climate at this locality is arid with mean annual rainfall ranging from 100 to 200 mm and limited almost exclusively to the summer months (December–February) . The soiCtenomys is still cursory. Numerous collected specimens, field studies and audio-recordings demonstrate that our study area is occupied by a single still unidentified Ctenomys species informally known as the Anillaco Tuco-Tuco. Although this species has been frequently reported as Ctenomys cf. knighti .Taxonomy in the genus  knighti  or C. af knighti , a formaWe recorded vocalizations of free-living individuals of the Anillaco Tuco-Tuco during the breeding seasons (June–September) of 2014 and 2015. The breeding season was identified on the base of captures of juveniles and reproductive females (pregnant and lactating) during five consecutive years . Recordings were made from outside the burrows in which animals vocalized using a Sennheiser ME-67 directional microphone mounted on a Rycote WS4 blimp windscreen and attached to a Marantz Professional PMD-661/MKII digital recorder. Sounds were recorded at a sample rate of 44.1 kHz and at 24 bit depth. The gain setting of the recorder was the same for all recordings. During recording sessions, J. Amaya positioned himself among a group of inhabited burrows and waited for Anillaco tuco-tucos to vocalize. When a vocalization was perceived he walked slowly and silently approaching to the sound source. Since LRVs are unpredictable and occur underground, none of the vocalizations recorded was complete . However, we took notes on how many series were missed, in order to characterize the composition of each bout , while lTo provide critical information regarding the sex and sexual maturation of animals whose vocalizations were recorded in the field, we captured 17 of the recorded wild individuals  using plastic tube traps set at the burrow entrance closest to where each animal had been recorded. Trapped animals were transported to the laboratory where each individual was weighed, sexed, and marked with a coded michrochip  inserted beneath the nape skin. To ascertain that the tuco-tuco trapped was indeed the one that was recorded we proceeded as follows. The mound where the animal was captured was left open, and each animal was kept for 24 h in the laboratory. If the capture mound was still open the next day at the time of release, we were confident that the individual recorded was the same one that was captured. If the mound was closed, then at least a second individual was inhabiting the burrow system, and no certainty was possible regarding the correspondence between the trapped individual and the one recorded; in this case, the vocalizations recorded at that burrow were discarded. In total, we captured and recorded 20 individuals but discarded recordings from three that could not be identified as producing the sounds recorded as follows: first, a male and then two females were captured in the same mound; second, a female and then a male were trapped in the same mound, and third, we captured a male but could not capture the other individual in the same burrow system.http://www.birds.cornell.edu/ raven) using the following spectrogram parameters , 3 dB Filter Bandwith: 135 Hz; Time grid–Overlap: 50%, Hop size: 256 samples (= 5.33 ms); Frequency grid–DFT size: 4,096 samples, Grid spacing: 11.7 Hz). All recordings were band-pass filtered between 80–5,000 Hz in Raven Pro 1.4 to eliminate sources of disturbance and distortion in central-tendency acoustic measurements. We manually delimited selection borders along the time axis in the waveform at points in which the waveform reached minimum energy values and changed its general repetitive pattern; this allowed us to delimit three minimum sound types in the vocalization  or below the peak amplitude of the waveform envelope (for time measurements) independently of amplitude differences in recordings of identical sounds that were recorded at different distances. However, since many of our recordings had a reduced signal-to-noise ratio we made our measurements comparable by obtaining peak frequency values and time and frequency central-tendency values for each selection, at the expense of loosing information contents. Peak frequency values are not influenced by the selection of the standardization threshold because by definition they are used to set the threshold. Central-tendency measurements are robust to differences in relative amplitude between sounds being compared and are based on internal cumulative-energy thresholds; results should be especially robust if energy is concentrated in a comparatively narrow band in which frequencies are similarly attenuated by the medium through which the sounds travel before being recorded. By using peak values and central-tendency measures, we circumvent the problem of making our measurements comparable in recordings with poor signal-to-noise ratio. We recognize that this implies losing the ability to obtain some comparable measures , but allows us to adequately characterize the signal of interest.To determine if production of vocal signals was influenced by body size in males, we used Pearson correlation tests (N = 14) to assess the relationship between the mean peak frequency of notes in triad series and individual notes and the weight of the male producing the call. Females were not included together with males in these analyses because of their significantly lower weight and mean peak frequencies, which might artificially result in a high correlation between weight and peak frequency by conflating the effects of sex and weight. Due to low sample size (n = 3) it was not possible to perform Pearson correlation tests separately in females.Larrea sp., Opuntia sp., and Parkinsonia praecox). Sounds were recorded using a Zoom H4n digital hand recorder system with built-in microphones (sample rate of 44.1 kHz and 16 bit depth) and the behavior of the animals was filmed simultaneously with a digital video-camera (Nikon D5100). Video and audio recordings of behavioral interactions allowed detailed characterizations of the contexts in which vocalizations were produced.To learn more regarding the behavioral contexts in which LRVs are produced we recorded vocal output during experimentally manipulated encounters. Five adult male and seven adult female Anillaco tuco-tucos were captured using plastic live traps as described above; after capture, the animals were transported to the Chronology Laboratory at CRILAR, where they were housed in an artificial burrow system consisting of four transparent glass enclosures with PVC tubes placed along the outer edges of each enclosure; the PVC tubes had been cut in half lengthwise to allow observations of the animals from outside of the experimental apparatus . Each enObservations and recordings began at least one week after animals had been placed in captivity, allowing acclimatization and territory establishment in the artificial burrow system. To observe and record the associated sounds produced we staged encounters between individuals that lasted 20–40 min; all encounters were conducted during June–September in 2014, 2015 and 2016. By opening and closing the doors within the enclosure we allowed contact between animals according to experimental requirements. The experimental subjects in 2014 were two males; male A (267 g) and male B (168 g) and two females; female C (140 g) and female D (160 g), in 2015 the same two males; male A (274 g) and male B (220), and two females; female C (160 g) and female E (110 g), and in 2016 three males; male F (144 g), male G (177 g) and male H (170 g) and four females; female I (165 g), female J (137), female K (133 g) and female L (125 g). We completed 97 encounters distributed in three encounter types: 1) male-male; 2) female-female and; 3) male-female encounters .We characterized the behavior of individuals during the encounters using the video recordings. We analyzed behavior in 19 male-male encounters , all five male-female and the single female-female encounters with LRVs.We analyzed 10 min per encounter that were selected based on the intensity of interactions and on the presence of LRVs. For each encounter, we measured an instantaneous sample point every 15 s (40 sampling points per encounter) at which we assessed whether each animal was performing any of three previously defined behaviors. In decreasing level of aggression, these behaviors were: 1) Aggression: individuals fighting or at a distance less than the length of the body while displaying aggressive behavior ; 2) Contact: individuals in contact or at a distance less than the length of the body without displaying aggressive behavior ; and 3) Independence: individuals at a distance greater than the body length . We calculated and graphed the percentage of samples per encounter in which each behavior occurred and compared how these proportions differed between the three different encounter types .To assess the effect of LRVs, we compared the behaviors exhibited in two 15-s sample points, one preceeding and one following the emission of vocalizations, using the previously defined behavioral categories of Aggression, Contact and Independence . We quanTo quantify the intensity and frequency of vocal output during encounters we defined and calculated the values of three variables: probability that a staged encounter would result in a LRV , number of vocalizations per staged encounter  and number of vocalizations VVE; total number of LRVs/staged encounters in which vocalizations occurred).All procedures followed the guidelines of the American Society of Mammalogists for the use of wild mammals in research . All expLong-range calls of Anillaco tuco-tucos were low-frequency, broad-band, loud, and long sounds that were composed of two syllable types: series and individual notes ; Table 1Series had two sound types: a) notes, which were the louder and lower-pitched sounds that initiated and finished a series, and b) soft-notes, which were the softer and higher-pitched sounds that were always sandwiched by notes . DependiMales were markedly heavier than females  they attempted to court each other, briefly giving courtship vocalizations. However, this lasted for a few moments and was followed by aggressive interactions. No courtship vocalization was observed during subsequent encounters. The fact that males briefly courted other males  during their first encounters, suggests that sexing between individuals requires from a close approach. The first male-male encounters seemed to result in sex recognition, indicating that subsequent encounters of non-naive animals were truly representative of male-male interactions. Thus, what might appear at first to represent a case of pseudoreplication, becomes an important tool that shows the need of previous contact between animals to ascertain their sex-specific behavior.In sum, males gave LRVs in proportionally more encounters and a greater number of times per encounter when facing another male than when facing a female, while females never gave LRVs in male-female encounters and did so only once in female-female encounters. While encounters between males were typically aggressive, male-female encounters appeared to result in courtship behavior.In this paper we have shown that long-range calls of Anillaco tuco-tucos are low-frequency, broad-band, loud, and long sounds composed by the repetition of two syllable types: series (formed by two to four notes and one to three soft-notes) and individual notes. All vocalizations were initiated with series, but not all had individual notes, and the arrangement and number of syllables was highly variable, without exhibiting any consistent pattern. These vocalizations were sexually dimorphic and were given mostly by males and seldom by females under natural conditions. Acoustic features were independent of body mass in males, while reduced sample size precluded this evaluation in females. Results from staged encounters of captive animals suggest that LRVs occur mostly among males and are associated primarily with agonistic contexts, but future studies using a larger number of individuals should determine the generality of this pattern.Ctenomys: Type I and Type II . Soft-notes differ from series notes in that they have different spectral parameters and have markedly lower relative energy values. It is important to emphasize that soft-notes are not simply reverberations from series notes. Specifically, a) we recorded some abnormal vocalizations in which the soft-notes occurred before the first note of a series, b) we recorded one series composed only of soft-notes, and c) soft-notes are not present in individual notes, as would be expected if the former were simply reverberations of the latter. Thus, we hypothesize that soft-notes are a distinctive but overlooked component of the long-distance calls of many Ctenomys species.The three types of series  found in the Anillaco Tuco-Tuco were complex structures composed by notes and soft-notes. Soft-notes have not been reported in the literature before, and their description represent another new aspect of vocal communication in ctenomyids. However, they seem to be present in several other um sensu consistsCtenomys, a necessary pre-requisite to support the idea that important information can be encoded in the rhythmic pattern within a species , and the variable number of individual notes created a diverse collection of syntactic patterns in vocalizations that would seem to provide the opportunity to encode multiple types of information within these calls. Here we provide the first evidence of substantial variation in the rhythmic patterns of LRVs in a single species of  species .Ctenomys.Vocal features such as peak frequency of series-notes and individual notes and syntactic patterns were sexually dimorphic, providing an additional dimension of variability in the long-distance vocalizations of ctenomyids. Future experimental studies designed to determine what types of information are conveyed in long-distance vocalizations and how that information is encoded will substantially improve our understanding of communication within the genus Ctenomys have generally been suggested to facilitate maintenance of individual territories due to their loudness  by males in our study population. Third, males produced LRVs with greater frequency during male-male encounters compared to male-female encounters. Finally, males in the study population appeared to have larger home ranges that were more spatially segregated than those of females , suggesting that males may have greater need for long-distance signals that advertise their presence in the habitat. Supporting this, female Anillaco Tuco-Tucos gave LRVs less often than males. Due to their apparent rarity, the function and acoustic features of LRVs in female tuco-tucos remain inadequately known.Future comparative studies of long-distance ctenomyids vocalizations will benefit from efforts to relate the acoustic features and emision of such vocalizations to the behavioral and ecological contexts in which they occur, and will shed light on the evolution of the complex vocal communication in this Neotropical radiation of fossorial rodents.Ctenomys sp.)Eight acoustic parameters measured to characterize the LRV of the Anillaco Tuco-Tuco (Peak Frequency (Hz): frequency at which the peak power occurs within the selection.IQR (Inter-quartile Range) Bandwidth (Hz): the difference between the 1st and 3rd Quartile Frequencies.Bandwith 90% (Hz): difference between the frequency 5 and 95% frequencies.1st Quartile Frequency (Hz): frequency that divides the selection into two intervals containing the 25 and 75% of the energy in the selection.3rd Quartile Frequency (Hz): frequency that divides the selection into two intervals containing the 75 and 25% of the energy in the selection.Frequency 5% (Hz): frequency that divides the selection into two frequency intervals containing the 5 and 95% of the energy in the selection.Frequency 95% (Hz): frequency that divides the selection into two frequency intervals containing the 95 and 5% of the energy in the selection.Duration 90% (ms): difference between the 5% time  and the 95% time .10.7717/peerj.2559/supp-1Supplemental Information 1Ctenomys sp.) recorded in the field. Each scheme is preceded by the number of individual, weight at time of the recording (in square brackets) and quantity of series without rec (dotted rectangle). Each scheme corresponds to a single recording. Wide gray boxes represent x number of series  and thin white boxes represent x number of individual note.Figure S1. Schematic representation of number of series and individual notes in the long-range vocalizations of the Anillaco Tuco-Tuco  was studied.Figure S2. Schematic representation of the artificial burrow system where behavioral context of long-range vocalizations of the Anillaco Tuco-Tuco  in captivity. Encounters with long-range vocalization (white background) and first male-male encounters with courtship vocalization (gray background). Bars indicate the percentage of occurrence of each behavior  per encounter.Figure S3. Behavior during staged vocal encounters of the Anillaco Tuco-Tuco  in laboratory experiments to assess the function of long-range vocalizations. Three types of encounters were performed: male-male, male-female and female-female. Capital letters indicate the identity of each individual used in each encounter type and numbers in parentheses indicats the number of staged encounters between the same individuals.Table S1. Design of staged encounters between individuals of the Anillaco Tuco-Tuco (Click here for additional data file.10.7717/peerj.2559/supp-5Supplemental Information 5Raw data.Click here for additional data file.10.7717/peerj.2559/supp-6Supplemental Information 6Audio sample of five diads of a single record.Click here for additional data file.10.7717/peerj.2559/supp-7Supplemental Information 7Audio sample of two triads of a single record.Click here for additional data file.10.7717/peerj.2559/supp-8Supplemental Information 8Audio sample of two tetrads of a single record.Click here for additional data file.10.7717/peerj.2559/supp-9Supplemental Information 9Audio sample of individuals notes of a single record.Click here for additional data file.