Augmented reality (AR) has the potential to support complex neurosurgical interventions by including visual information seamlessly. This study examines intraoperative visualization parameters and clinical impact of AR in brain tumor surgery.n = 39) or conventional neuronavigation (n = 16) after randomization, have been included prospectively. Surgical resection time, duration/type/mode of AR, displayed objects , pointer-based navigation checks (n), usability of control, quality indicators, and overall surgical usefulness of AR have been assessed.Fifty-five intracranial lesions, operated either with AR-navigated microscope (P < 0.001).AR display has been used in 44.4% of resection time. Predominant AR type was navigation view (75.7%), followed by target volumes (20.1%). Predominant AR mode was picture-in-picture (PiP) (72.5%), followed by 23.3% overlay display. In 43.6% of cases, vision of important anatomical structures has been partially or entirely blocked by AR information. A total of 7.7% of cases used MRI navigation only, 30.8% used one, 23.1% used two, and 38.5% used three or more object segmentations in AR navigation. A total of 66.7% of surgeons found AR visualization helpful in the individual surgical case. AR depth information and accuracy have been rated acceptable . The mean utilization of the navigation pointer was 2.6 × /resection hour (AR) vs. 9.7 × /resection hour (neuronavigation); navigation effort was significantly reduced in AR  provides the highest usability while blocking the operative field less frequently. Visualization quality will benefit from improvements in registration accuracy and depth impression.DRKS00016955.The online version contains supplementary material available at 10.1007/s00701-021-05045-1. The increase of visual information provided during neurosurgical procedures poses the threat of unwanted interference and cognitive overload for the surgeon. While the history of augmented reality (AR) visualization in neurosurgery already began in 1986 , 39, 42,In order to assess both the qualitative dimensions of the AR visualization at a given point during surgery and the quantifiable parameters indicating its overall usefulness, we applied a mixed methods study design involving direct participant observation in the operating room (OR) using a checklist specifically designed for this purpose, analysis of clinical data and microscope recordings, complemented by user interviews.b value=1300 s/mm2 with 60 directions per shell. The scans were performed with a standard ep2d sequence, for an acquisition time of 5:47 min. The participating surgeons received a two-stage (basic and case-based) training in HUD control and microscope navigation. They performed segmentation of tumors and additional anatomical structures themselves, usually on the day of surgery; tractography and import of nTMS-positive spots, where applicable, were provided in advance by the same lab members who routinely transfer preoperative brain mapping results into the surgical planning software, and were reviewed by the surgeons in the final version of the plan. All surgical cases have been performed in a regular OR environment using a standard surgical microscope  and the latest update of a commercial software for cranial planning and navigation . Preset HUD views enabled by the microscope navigation software include augmented volumes/outlines of a target and probe’s eye  both as overlay and picture-in-picture (PiP), as well as navigation view in axial, coronal, and sagittal (ACS) orientation (PiP only). An illustrated case marks the combined view of target volumes and navigation PiP , diffusion tensor imaging (DTI), and brain mapping results from navigated transcranial magnetic stimulation (nTMS). The structural acquisitions on a 3 T MRI scanner  included a T1 MPRAGE anatomical sequence 0.9 mm isotropic resolution, TR/TE 2300/2.32 ms, TI 900 ms, and flip angle 8 degree, for an acquisition time of 5:18 min. Diffusion data for tractography included 2 mm isotropic resolution whole brain acquisitions, TR/TE 7500/95 ms, 1 shell n=92) with compatible imaging data, operated either with the AR-navigated microscope or conventional neuronavigation, has been analyzed prospectively. Cases have been assigned randomly to one of the groups unless the procedure times of included AR cases overlapped; with only one AR setup being available, subsequent cases if occurring had to be assigned to the control group (pseudorandomization). All cases with technical failures occurring at the beginning of surgery, with incomplete documentation and/or missing or corrupted microscope video, have been excluded from analysis . Technical issues were the most important reason for exclusion of cases in the AR group, while incomplete data caused most case exclusions in the control group. The screen-based conventional neuronavigation system was available in all cases as a backup solution. Surgical resection time, duration/type/mode of AR, displayed objects , pointer-based navigation checks (n), and case-specific visual quality indicators  have been assessed based on participant observation in the OR and video analysis of the microscope recording (see Table n=7) on a 5-point Likert scale  during and after the intervention. Ethical approval has been obtained from the local IRB , and written patient consent was collected prior to data acquisition. This study has been registered with the German Clinical Trials Register (DRKS00016955).The main inclusion criterion for study cases was that there had to be expected added benefit from navigation information during at least one step of surgery . Between November 2017 and September 2018, a series of navigated brain tumor cases , color coding, shading, translucence, and occlusion, contrast, type of  data, transparency of data source , type of view, number, position and complexity of displayed objects, overlay accuracy, coherence of image fusion and integration of contextual information, intuitivity and comprehensibility of AR scene, and relevance for surgical decision-making during intervention . After correction for multiple testing using the Bonferroni test, two-tailed probability values of n=39, conventional neuronavigation n=16). Mean patient age was 48.1 year ; 54.5% were male with a m/f ratio of 1.2:1. The lesions included 96.4% tumor and 3.6% vascular cases . Dominant neuro-oncological lesions were 38.2% glioblastoma (GBM), 14.5% anaplastic astrocytoma (aAST), 9.1% metastasis (Met), 7.3% anaplastic oligodendroglioma (aODG), 7.3% oligodendroglioma (ODG), and 23.4% other, of which 29.1% were recurrences  had a transient deficit after surgery , 12.7% of patients  had a permanent new deficit after 3 months , and 9.1% of patients  improved postoperatively .A total of 14.5% of patients  surgical time per case. We could not identify any surgical complications related to HUD-based AR utilization.In the AR group, AR has been utilized in 44.4% mean 32.2 min) of total resection time (mean 72.5 min). In 43.6% of cases, important anatomical structures have been partially or completely obscured by AR information at least once during surgery. Frequently used HUD display modes were navigation view (75.7%) and target volume mode (20.1%), displayed predominantly as PiP (72.5%) and occasionally as overlay (23.3%), rarely as a combination of both (4.2%) . Additional tumor segmentation was the most frequent object type in both groups (84.6% in AR vs. 62.5% in navigation), followed by tractography for motor and language (56.4% in AR vs. 56.2% in navigation), nTMS data for cortical hotspots (38.5% in AR vs. 6.2% in navigation), and anatomical structures such as vessels, cranial nerves, or ventricles (12.8% in AR vs. 12.5% in navigation). MRI-only navigation view has been utilized in 7.7% (AR) and 18.8% of cases , respectively   have been displayed in the AR group compared to 1.4 objects in the conventional neuronavigation group 0–3; SD 1)  compared to conventional neuronavigation (n=12). Notable differences were found in spatial understanding of information , visual accuracy of overlay , visual transparency of data source , and visual comprehensibility . In comparison, coherence of image fusion  and relevance of visualization  showed no significant differences.The dimensions of visualization quality have been rated equally to poorly in the navigated microscope (n=9), offset range in AR visualization was 0–7 mm. A total of 66.7% of participants stated that AR visualization was helpful for their surgical case; 76.9% of those cases were deep-seated lesions. The surgeons who found AR visualization helpful displayed two navigation objects on average and showed in 84.6% a fine command of the HUD visualization. A total of 65.4% of them indicated that their focus view remained unblocked during surgery, using navigation view as PiP display in 88.6% of the cases, while 30.8% encountered technical issues during preparation and/or surgery. The mean pointer utilization was reduced in this subgroup (2.2×/resection hour), whereas the mean AR utilization was increased (39.5 min).When verified with the navigation pointer and a sterile paper ruler by the surgeon  and the visual integration in the field of view  , namely, during the targeting of small structures, the consulting of tumor borders, or the identification of functionally eloquent areas in conjunction with intraoperative neuromonitoring. While both neuro-oncological and neurovascular cases are frequently reported in the literature as predominant application areas, the majority of our cases turned out to be intra-axial tumors. However strongly articulated as a clinical demand, the support of neurovascular cases using the existing software application was restricted by prevailing technical limitations for the duration of this study. Here, future solutions need to incorporate the detailed vascular architecture surrounding the surgical target , preferably containing information on blood flow  and oxygen concentration .In several studies, AR is frequently used for preoperative craniotomy as well as skin incision planning and stated as helpful or beneficial , 20, 33 In general, AR visualization in the microscope HUD can help account for registration inaccuracy and brainshift , 22 usinIn the minimal version of HUD-based visualization, the navigated microscope constitutes an integrated pointer without exploiting the potential of an AR display. As has been discussed before, AR visualization can contribute to focused information flow during surgery, reducing alternate viewing directions and attention shifts , 30 as wTo date, there are no established standards for measuring the quality of AR visualization in neurosurgery. Two publications suggest a strategical framework for device assessment focusing on technical setup, display types, and the processing of imaging data , 35. BasThe selection of appropriate information depends on several contributing factors, such as availability  and individual command of software functionality and personal habits. We observed no difference in the type of data used for intraoperative visualization between AR guidance and conventional neuronavigation. Accordingly, AR visualization quality largely depends on the quality of the underlying data , usuallyDespite being the favored view in our study, the AR navigation view does not reveal context information outside the currently displayed sectional planes; it requires further interaction, such as change of focal plane, to provide visualized information above and below the actual resection plane, which can be crucial in minimally invasive or keyhole approaches .n>3) cannot be recommended as they cause distraction and informational overload . Regrettably, current commercial AR visualization of volumes still remains restricted by at least a few unsolved issues in transparency, shading, and visual occlusion, preventing a more effective merging of physical and virtual information. Eliminating the necessity of a multiple screen scenario during consultation of presurgical planning data, they can best be used as a recapitulatory step before switching to more instructive views . With the ongoing technical improvements in digital visualization, we expect a further and profound change towards better integrated AR information that can be used comfortably throughout surgery at different scales and degree of detail.While the current software generation introduced more naturalistic 3D volume injection in navigated microscopes, any conveyed object depth information  has been yet rated unintuitive by the surgeons. As the visualization remains always on top of the AR scene, the placement of objects does not follow context or background features, and volumetric data does not properly merge with the scene . ConsequAs we encountered a comparably high number of technical issues  in the course of this study, we suggest implementing better user guidance in the software interface for AR navigation. Additionally, built-in features for measuring registration error and accuracy of overlay  would beA major limitation of this study is the imbalance of the two groups after randomization, which can be largely explained by not using a 1:1 allocation at the beginning followed by different reasons for subsequent case exclusion .Navigating the focal point of the surgical microscope does not exploit the full potential of AR visualization in surgery. Microscope navigation is one case study among many which can contribute to the understanding of intraoperative visualization requirements.Most cases of our series were intracranial tumors. We included only two neurovascular pathologies  for the majority of those cases at our institution is usually treated relying on anatomical landmarks (without neuronavigation) and/or intraoperative imaging. Particularly in AVMs, we expect considerable improvements with an upcoming software update allowing for better discrimination of arterial feeders, venous drainage, and nidus structures based on high-resolution three-dimensional, partially automated vessel segmentation. A randomized study with high case numbers will be needed for validation of presented findings; however, this might prove difficult as it involves abandoning the established gold standard of neuronavigation in potentially complex procedures with users less experienced in AR navigation. Accordingly, there are no prospective studies showing a significant difference between AR-aided surgeries versus navigation-guided procedures regarding morbidity, mortality, and clinical effectiveness . Given the differences in group size and distribution of pathologies, the clinical results of this study can only be suggestive in regard of certain tendencies. A considerable limitation of current AR navigation using visual overlay is the visualization offset, which can be even more distracting at high magnification than comparable offset in pointer-based neuronavigation. Here, a standardized investigation across technical setups, different pathologies, and surgical approaches will be necessary. As for the investigation of intraoperative visualization standards, a multicenter comparison could provide further insights in terms of applicability and scalability of requirements and recommendations. Future work should include the visual standardization across applications in particular in order to contribute substantial clinical data to the ongoing assessment of augmented, mixed, and virtual reality techniques in neurosurgery .The evaluation of AR in exoscopic surgery is subject of a follow-up study at our institution.HUD-based AR visualization in routine brain tumor surgery is clinically feasible and safe. Its most salient feature is the pointer-free navigation during tissue preparation and tumor removal, minimizing the current ergonomic hindrances, which require surgeons to look alternately at multiple displays. While the technical workflow is at large compliant with daily surgical routine, the visualization quality still impacts surgical cognitive load and performance. The new software generation offers a more “realistic” yet clearly distinguishable style of visualization for surgically relevant information. Known restrictions of the technology are due to, firstly, the overlay of working and viewing area; secondly, the lack of stereoscopic three-dimensional depth information in the AR scene; and thirdly, potential visualization offset caused by MRI data resolution, registration errors, and brainshift during surgery. Factors promoting the application of AR navigation, as identified in this study, are deep-seated lesions and peripheral navigation view including two displayed essential objects  in a trained user. Personal surgical preferences affect the utilization of the technique; since the navigated microscope is an extension of conventional neuronavigation, the habits of use and visualization style are largely comparable.Supplementary file1 (DOCX 16 KB)Supplementary file2 (DOC 37 KB)Supplementary file3 (DOC 221 KB)Below is the link to the electronic supplementary material.


S)- and (R)-PF74 and further enrichment of the samples (≥98%) using chiral HPLC resolution. The biological activities of each enantiomer were then evaluated, which determined (S)-PF74 (IC50 1.5 µM) to be significantly more active than (R)-PF74 (IC50 19 µM). Computational docking studies were then conducted to rationalise this large discrepancy in activity, which indicated different binding conformations for each enantiomer. The binding energy of the conformation adopted by the more active (S)-PF74  was calculated to be more favourable than the conformation adopted by the less active (R)-enantiomer  in agreement with experimental observations.PF74 is a capsid-targeting inhibitor of HIV replication that effectively perturbs the highly sensitive viral uncoating process. A lack of information regarding the optical purity (enantiomeric excess) of the single stereogenic centre of PF74 has resulted in ambiguity as to the potency of different samples of this compound. Herein is described the synthesis of enantiomerically enriched ( Human Immunodeficiency Virus (HIV) is a pathogenic retrovirus that infects cells of the human immune system and, if left untreated, can result in Acquired Immunodeficiency Syndrome (AIDS). An estimated 38 million people are infected worldwide and, since the epidemic began in the early 1980s, approximately 33 million people have died from HIV-related deaths . CombinaThe HIV-1 capsid protein (CA) multimerizes to form a fullerene-like shell that encapsidates the viral RNA genome and is essential for both early and late stages of the viral replication cycle . Small mPF74 is a non-natural derivative of phenylalanine, a sub-structure that has been further optimised in lenacapavir. However, crucial information regarding the stereogenic centre present in PF74 is scarce in published syntheses and studies on the biological activities of this compound, and is not readily available from commercial suppliers of PF74 ,12,13,14The scope of this project was to study stereochemical integrity in the synthesis of PF74, and evaluate the biological activity of the two enantiomers in both cell assay and through computational docking.S)-, (R)- and racemic PF74 using enantiomerically enriched starting materials, which allowed us to assess any epimerisation and to compare our synthetic material with commercially available PF74. A PyBop-mediated amide coupling between 2-methylindole-3-acetic acid (1) and either (S)-, (R)- or racemic phenylalanine methyl ester hydrochloride  was utilised to generate the amide containing methyl ester 3  with N-methylaniline generated PF74 .Our improved synthesis of PF74 was conducted based on a previously reported literature synthesis, with some optimisation . Due to  ester 3 . Yields 4 + m/z 373.1523, found m/z 373.1514. IR (thin film) 2952, 1741, 1651.HRMS (ESI) exact mass calculated for Cd-phenylalaninateMethyl (2-(2-Methyl-1H-indol-3-yl)acetyl)- (3b)3b was obtained as a viscous oil. Following the general procedure outlined above, 719 mg (78%) of compound 1H NMR  δ 7.92 , 7.42 , 7.31 , 7.18 , 7.13 , 7.09 , 6.09 , 6.70 , 6.00 , 4.90–4.76 , 3.64 , 3.64 , 3.03–2.85 , 2.28 . 13C NMR  δ 171.9 (-CONH), 171.1 (-COOR), 135.6 (Ar-C), 135.5 (Ar-C), 133.4 (Ar-C), 129.1 (2 × Ar-CH), 128.5 (2 × Ar-CH), 128.4 (Ar-C), 127.0 (Ar-CH), 121.9 (Ar-CH), 120.2 (Ar-CH), 118.0 (Ar-CH), 110.5 (Ar-CH), 104.6 (Ar-C), 52.8 (-CH), 52.3 (-OCH3), 37.7 (-CH2), 32.1 (-CH2), 11.6 (-CH3). 21H22N2O3Na [M + Na]+ m/z 373.1523, found m/z 373.1514. IR (thin film) 2952, 1741, 1651.HRMS (ESI) exact mass calculated for Cdl-phenylalaninateMethyl (2-(2-Methyl-1H-indol-3-yl)acetyl)- (3c)3c was obtained.Following the general procedure described above, 925 mg (quantitative) of 1H NMR  δ 7.89 , 7.42 , 7.31 , 7.18 , 7.15–7.11 , 7.11–7.05 , 7.00 , 6.70 , 5.99 , 4.88–4.80 , 3.64 , 3.64 , 3.00–2.86 , 2.28 . 13C NMR  δ 171.9 (-CONH), 171.1 (-COOR), 135.6 (Ar-C), 135.5 (Ar-C), 133.4 (Ar-C), 129.2 (2 × Ar-CH), 128.5 (2 × Ar-CH), 128.4 (Ar-C), 127.0 (Ar-CH), 121.9 (Ar-CH), 120.2 (Ar-CH), 118.0 (Ar-CH), 110.5 (Ar-CH), 104.7 (Ar-C), 52.8 (-CH), 52.3 (-OCH3), 37.7 (-CH2), 32.2 (-CH2), 11.6 (-CH3). 21H22N2O3Na [M + Na]+ m/z 373.1523, found m/z 373.1514. IR (thin film) 2952, 1741, 1651. Melting point 126–130 °C.HRMS (ESI) exact mass calculated for C3 (1 equiv) in tetrahydrofuran (0.23 M) and water (0.34 M) was added a solution of sodium hydroxide (1.5 equiv) in methanol (1 M NaOH) and the resulting solution was stirred vigorously at room temperature for 18 h. The reaction mixture was diluted with water and the crude biphasic mixture was washed with dichloromethane to remove organic impurities. The aqueous phase was then acidified with 2 M aqueous hydrochloric acid forming a thick white precipitate. The aqueous phase was extracted with dichloromethane and the combined organic phases were dried with magnesium sulfate, filtered, and concentrated in vacuo. The compound was used without further purification.To a stirred solution of d-phenylalanine(2-(2-Methyl-1H-indol-3-yl)acetyl)- (4a)4a was obtained. Following the general procedure described above, 989 mg (97%) of 1H NMR  δ 8.14 , 7.34 , 7.26 , 7.16 , 7.09 , 7.09 , 6.99 , 6.74 , 6.15 , 4.76 , 3.61 , 3.00 , 2.89 , 2.13 . 13C NMR  δ = 174.0 (-COOH), 172.9 (-CONH), 135.5 (Ar-C), 135.4 (Ar-C), 133.8 (Ar-C), 129.2 (2 × Ar-CH), 128.6 (2 × Ar-CH), 128.2 (Ar-C), 127.0 (Ar-CH), 121.8 (Ar-CH), 120.1 (Ar-CH), 117.8 (Ar-CH), 110.7 (Ar-CH), 103.7 (Ar-C), 53.2 (-CH), 37.0 (-CH2), 31.8 (-CH2), 11.4 (-CH3). 20H20N2O3Na [M + Na]+ m/z 359.1366, found m/z 359.1355. IR (thin film) 2916, 1724, 1618.HRMS (ESI) exact mass calculated for C(2-(2-Methyl-1H-indol-3-yl)acetyl)-l-phenylalanine (4b)4b was obtained as a solid. Following the general procedure outlined above, 358 mg (74%) of 1H NMR  δ 7.89 , 7.36–7.29 , 7.18 , 7.13 , 7.11 , 7.07–7.00 , 6.75 , 5.99 , 4.69 , 3.64 , 3.04 , 2.94 , 2.20 . 13C NMR  δ = 174.0 (-COOH), 172.9 (-CONH), 135.5 (Ar-C), 135.4 (Ar-C), 133.8 (Ar-C), 129.2 (2 × Ar-CH), 128.6 (2 × Ar-CH), 128.2 (Ar-C), 127.0 (Ar-CH), 121.8 (Ar-CH), 120.1 (Ar-CH), 117.8 (Ar-CH), 110.7 (Ar-CH), 103.7 (Ar-C), 53.2 (-CH), 37.0 (-CH2), 31.8 (-CH2), 11.4 (-CH3). 20H20N2O3Na [M + Na]+ m/z 359.1366, found m/z 359.1355. IR (thin film) 2916, 1724, 1618. Melting point 140–143 °C.HRMS (ESI) exact mass calculated for Cd-phenylalanine(2-(2-Methyl-1H-indol-3-yl)acetyl)- (4c)4c was obtained.Following the general procedure outlined above, 491 mg (quantitative) of 4a.Spectroscopic data matched that of acid 4  in N,N-dimethylformamide (0.3 mL) was added N-methylaniline , HATU  and N,N-diisopropylethylamine  and the resulting solution was heated to 65 °C and stirred for 48 h. To the reaction mixture was added dichloromethane and half saturated aqueous sodium hydrogen carbonate solution and the aqueous phase was extracted with dichloromethane. The combined organic phases were washed with 5% aqueous lithium chloride solution, dried over magnesium sulfate, filtered, and concentrated in vacuo. The crude mixture was purified by column chromatography on silica gel using an eluent of 1–5% ethanol in dichloromethane.PF74 was prepared according to a modified literature procedure . To a stNatural (S)-PF74 (5a)5a was obtained.Following the general procedure described above, 63 mg (49%) of 1H NMR  δ 8.26 , 7.39–7.31 , 7.26 , 7.18–7.04 , 6.97–6.89 , 6.71–6.65 , 6.22 , 4.81 , 3.59 , 3.58 , 3.17 , 2.74 , 2.54 , 2.26 . 13C NMR  δ 171.4 (-CONR2), 170.9 (-CONH), 142.6 (Ar-C), 136.2 (Ar-C), 135.5 (2 × Ar-C), 129.9 (2 × Ar-CH), 129.3 (2 × Ar-CH), 128.4 , 128.2 (Ar-CH), 127.5 (2 × Ar-CH), 126.8 (Ar-CH), 121.6 (Ar-CH), 119.9 (Ar-CH), 117.9 (Ar-CH), 110.6 (Ar-CH), 104.6 (Ar-C), 51.2 (-CH), 38.9 (-CH2), 37.8 (-NCH3), 32.3 (-COCH2), 11.7 (-CH3). 27H27N3O2Na [M + Na]+ m/z 448.1995, found m/z 448.1977. IR (thin film) 2924, 1638, 1595. HRMS (ESI) exact mass calculated for Ci-PrOH in n-hexane, 1 mL min−1, λ 208 nm) tRnatural = 32.511 min (54.8%), tRunnatural = 40.831 min (45.2%). + m/z 448.1995, found m/z 448.1977. IR (thin film) 2924, 1638, 1595. HRMS (ESI) exact mass calculated for Ci-PrOH in n-hexane, 1 mLmin−1, λ 208 nm) tRnatural = 32.550 min (35.0%), tRunnatural = 40.678 min (65.0%). + m/z 448.1995, found m/z 448.1977. IR (thin film) 2924, 1638, 1595. HRMS (ESI) exact mass calculated for Ci-PrOH in n-hexane, 1 ML min−1, λ 208 nm) tRnatural = 32.227 min (50.4%), tRunnatural = 40.120 min (49.6%). [αc = 1.00, CHCl3).HPLC  and were cultured in DMEM (Dulbecco’s modified Eagle’s medium) with 10% FCS  and 10 μg/mL gentamicin. The VSV-G pseudotyped single-cycle HIV-1 reporter lentiviral vector CSGW is self-inactivating, and encodes GFP expressed from an internal spleen focus-forming virus (SFFV) long terminal repeat (LTR) , packageCells were seeded in 96-well plates  immediately prior to infection or treatment. Briefly, suspension HEK293T cells were infected with a titrated challenge of serially diluted CSGW-containing supernatant. At 48 h post infection, cells were trypsinized and incubated at 37 °C to enable a single cell suspension and fixed . The fixed cells were incubated to allow complete virus inactivation prior to analysis. Quantification of GFP positive cells (representative of GFP-reporter CSGW infection) was carried out using flow cytometry with 10,000 events acquired per well (Guava EasyCyte cytometer). From the titration performed with CSGW, the concentration of virus-containing supernatant required to produce 15–20% infection of cells was calculated. This dilution was then used in in fixed CSGW dose assays.For fixed compound dose assays, a CSGW titration as described above was performed with one exception. The final addition of 50 µL fresh media described above was substituted with the addition of 50 µL of a 4% DMSO solution of compound  in media. The plates were then incubated for 48 h before being fixed  and analysed by flow cytometry.m (×4 dilution series). In addition to the compound, each well contained 100 µL of a suspension of HEK293T cells in fresh media  and 50 µL of diluted CSGW  in fresh media with a final concentration of 1% DMSO per well. The plates were then incubated for 48 hours before being fixed  and analysed by flow cytometry.For compound dose response assays, each compound was titrated across a 96-well plate to give a 50 µL solution with a maximum final concentration of 100 µM to a minimum final concentration of 95 pAll structure preparation and docking was performed using the Schrödinger suite (build 2019-1) The enantiomers of PF74 were drawn with ChemDraw and converted to a 3D structure and energy minimized using the OPLS3e forcefield in LigPrep . The hexameric quaternary structure of capsid PDB: 2XDE  was geneS)-, (R)- and racemic PF74 were based on a previously published procedure [S)- and (R)-PF74. Evaluation of the biological activities was conducted in HEK293T cells to assess the ability of each enantiomer to inhibit the infectivity of the HIV viral vector CSGW-VSV-G. In broad agreement with recently published work [S)-PF74 to be significantly more active (IC50 = 1.5 µM) than (R)-PF74 (IC50 = 19 µM). In an effort to rationalise this observation, docking studies were conducted which showed the two enantiomers adopted different conformations in the PF74 binding site. The more active (S)-PF74 had additional favourable interactions compared to (R)-PF74, which was reflected in the calculated binding energies of each conformation, the more active (S)-PF74  being significantly lower in energy than (R)-PF74 .To summarise, our improved syntheses of (rocedure . Chiral hed work , this te


Kaposi’s sarcoma-associated herpesvirus (KSHV) preferentially infects and causes Kaposi’s sarcoma (KS) in male patients. However, the biological mechanisms are largely unknown. This study was novel in confirming the extensive nuclear distribution of the androgen receptor (AR) and its co-localization with viral oncoprotein of latency-associated nuclear antigen in KS lesions, indicating a transcription way of AR in KS pathogenesis. The endogenous AR was also remarkably higher in KSHV-positive B cells than in KSHV-negative cells and responded to the ligand treatment of 5α-dihydrotestosterone (DHT), the agonist of AR. Then, the anti-AR antibody-based chromatin immunoprecipitation (ChIP)-associated sequencing was used to identify the target viral genes of AR, revealing that the AR bound to multiple regions of lytic genes in the KSHV genome. The highest peak was enriched in the core promoter sequence of polyadenylated nuclear RNA (PAN), and the physical interaction was verified by ChIP–polymerase chain reaction (PCR) and the electrophoretic mobility shift assay (EMSA). Consistently, male steroid treatment significantly transactivated the promoter activity of PAN in luciferase reporter assay, consequently leading to extensive lytic gene expression and KSHV production as determined by real-time quantitative PCR, and the deletion of nuclear localization signals of AR resulted in the loss of nuclear transport and transcriptional activity in the presence of androgen and thus impaired the expression of PAN RNA. Oncogenically, this study identified that the AR was a functional prerequisite for cell invasion, especially under the context of KSHV reactivation, through hijacking the PAN as a critical effector. Taken together, a novel mechanism from male sex steroids to viral noncoding RNA was identified, which might provide a clue to understanding the male propensity in KS. Although the incidence of Kaposi’ sarcoma (KS) is higher in men, little is known about the mechanisms by which male sex steroids contribute to this disparity. The present study confirmed the striking expression of the androgen receptor (AR) and its concordant nuclear distribution in KS tissues. High-throughput chromatin immunoprecipitation sequencing analysis showed that the AR had extensive binding sites in the KSHV genome, in which the highest enriched gene was PAN. PAN also exhibited the strongest upregulation of promoter activity and RNA transcription among various KSHV lytic genes after the male hormone treatment. Specifically, the effect was a result of the DNA-binding capability of AR to PAN promoter. Moreover, the AR induced dramatic cell invasion, especially under KSHV lytic replication, and the effect was greatly impaired by the inhibitory effect of siRNA on PAN RNA. This study provided a unique insight into the reason why KS occurred predominantly in men. Kaposi’s sarcoma (KS) occurs predominantly in men in the classic type or endemic population, and the male-to-female ratio ranges from 10:1 to 15:1 . The malIt was once recognized that the latency in the biphasic life cycle of KSHV was majorly responsible for KS oncogenesis until highly active antiretroviral therapy implementation targeting KSHV lytic infection dramatically declined AIDS-KS incidence, indicating the pathological role of virus reactivation in KSHV oncogenesis –20. In lTo our knowledge, this was the first study to identify a striking expression of AR in the nuclei of KS spindle cells at a very high level, compared with the sporadic cytoplasm distribution from control tissues. As a notable steroid hormone receptor of the nuclear receptor family, the AR is classically thought to function as transcriptional regulators in the nucleus. Upon ligand binding, the intracellular AR translocates to the nucleus and then engages with specific androgen-responsive elements (AREs) to modulate gene transcription . TherefoUsing ChIP-seq analysis, extensive binding sites of AR to the KSHV genome of several lytic genes were observed, and PAN exhibited the highest enrichment. We further determined the direct interaction between AR and PAN promoter by electrophoretic mobility shift assay (EMSA) assay using the recombinant protein of AR mutant, whose DNA-binding domain was deleted. Functionally speaking, male steroid hormone treatment led to significantly increased KSHV lytic gene expression, especially PAN, and consequently the infectious progeny production, as determined by luciferase reporter assay and quantitative real-time polymerase chain reaction (qRT-PCR). Mechanically speaking, it was the ligand-activated nuclear transport of AR that determines the successful expression of downstream PAN RNA. Finally, the tumorigenic capability of AR overexpression was most significantly impaired by RNA interference of PAN upon KSHV lytic replication in the cell invasion assay. Taken together, the results of our study indicated that male hormones were functionally involved in the male propensity of KS by serving as a canonical transcription factor to viral noncoding RNA PAN.All experiments in the present study were conducted according to the principles mentioned in the Declaration of Helsinki. The use of the clinical sections of classic KS tissue specimens, normal skin, and non-KS angioma tissues was reviewed and approved by the Institutional Ethics Committee of the First Teaching Hospital of Xinjiang Medical University . Written informed consent was obtained from all participants, and all samples were anonymized. All tissue specimens had a pathological diagnosis except normal skin samples. All samples were collected from Xinjiang province, northwestern China.primary effusion lymphoma cells , BC3, and JSC1  were generously provided by Dr. Erle S Robertson  and maintained in Roswell Park Memorial Institute 1640 medium supplemented with 10% FBS (HyClone). Androgen-sensitive human prostate adenocarcinoma cells (LNCap) (TCHu173) and androgen-independent prostate cancer cells (PC3) (TCHu158) were purchased from cell bank/stem cell bank of Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences . Charcoal-stripped fetal bovine serum (CD-FBS) (Sigma), from which endogenous hormones and growth factors were removed, was used for cell culture before DHT treatment.KS-derived endothelial SLK cells and 293T cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum . A derivative of SLK (termed iSLK) expressing a doxycycline-inducible replication and transcription activator (RTA) transgene was latently infected with a recombinant KSHV.219 virus, referred to as iSLK.219 cells, which constitutively expressed puromycin N-acetyl-transferase and green fluorescent protein (GFP); red fluorescent protein (RFP) was expressed during lytic replication . The iSLThe antibodies and reagents used were as follows: anti-AR antibody , anti-latency-associated nuclear antigen (LANA) monoclonal antibody , anti-immunoglobulin G (IgG) antibody , anti-hemagglutinin (HA) antibody , anti-Flag antibody , and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody . The secondary antibodies (Thermo Fisher Scientific) included goat anti-rabbit antibodies conjugated with Alexa Fluor 488 [A-11094], 555 [A27017], and 680 [A27020]), and goat anti-mouse antibodies conjugated with Alexa Fluor 488 [A-11001], 555 [A-21422], and 680 [A-28183]). DHT , protease inhibitor cocktail set III , phosphatase inhibitor cocktail , doxycycline hyclate , hygromycin , puromycin , and G418 disulfate salt  were used. Control siRNA (fluorescein isothiocyanate conjugate)-A , AR siRNA , Lipofectamine 3000 , 4’,6-diamidino-2-phenylindole (DAPI) , anti-Flag M2 affinity gel , recombinant protein A/G agarose , glutathione sepharose 4B , SYBR Green Master Mix Kit , Corning Transwell polycarbonate membrane cell culture inserts , collagen type I cell ware coverslips , Dual-Luciferase Reporter Assay System , and Cell Counting Kit 8  were also used in the study.Plasmids: Flag-tagged full-length AR  and its truncated constructs  were constructed by cloning the corresponding fragments into the pCDH-CMV-IRES-sf3-blast vectors . The glutathione S-transferase (GST)-fused truncated constructs of AR  were obtained by cloning the corresponding fragments into the pGEX-4T-1 vector. The reporter plasmid pGL3.0-RTAp, pGL3.0-LANAp, and pGL3.0-PANp were described previously ,29. A seTwelve KS tissues, one normal skin tissue, and six non-KS angioma tissues were collected. All the samples were formalin-fixed, paraffin-embedded, and immunostained with anti-AR or anti-LANA antibodies at a ratio of 1:200 as previously described . The resThe cell lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer  containing protease and phosphatase inhibitors. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes for immunoblotting with the indicated antibodies.The cells were fixed with 4% paraformaldehyde for 30 min at room temperature, permeabilized with 0.5% Triton X-100, blocked with 20% normal goat serum (Life Technologies), and reacted with indicated antibodies followed by fluorescent dye-conjugated secondary antibodies (1:1000). The dilution factor for individual primary antibodies was generally 1:200. The coverslips were mounted with an anti-fade mounting medium (Beyotime) and photographed using a digital camera and software (Olympus FV-1200).2, 0.2% Triton X-100, 1mM dithiothreitol, 0.5mM EDTA, and 0.2mM phenylmethylsulfonyl fluoride (PMSF) for 10 min at 4°C. The extracted nuclei were pelleted by centrifugation at 1300g for 5 min. The nuclei were lysed in sodium dodecyl sulfate (SDS) lysis buffer  for 10 min on ice. The lysates were subjected to sonication to obtain 200- to 500-bp fragments of DNA  and then centrifuged at 12,000 rpm at 4°C for 10 min to obtain the supernatants. The samples were precleared with pretreated protein A or G beads  for 2 h at 4°C. Then, 5% of the supernatants were kept as the input, and the remainder was divided into groups according to the experiment. The aliquots were incubated with pretreated protein A or G beads and 5 μg of anti-AR antibody or control rabbit immunoglobulin IgG antibody overnight at 4°C. After extensive washing with RIPA buffer, wash buffer , and Tris-EDTA (TE) buffer  (four times each), the beads were resuspended in TE buffer. The resuspended beads were subjected to RNase A and proteinase K digestion, and the crosslinking was reversed at 65°C for 8–10 h. DNA was recycled with a DNA purification kit . The primer sequences are summarized in The ChIP protocol was adapted from the Rockland website with some modifications . The cel7 iSLK.219 cells were harvested for the experiment. The lysates were sonicated to obtain DNA fragments of ~200 bp. The enriched DNA was subjected to library preparation for high-throughput sequencing. The ChIP-seq library was prepared according to Illumina instructions. The size selection range for the library was 100–400 bp.The procedures for ChIP for sequencing (ChIP-seq) were similar to those for ChIP described earlier, except for the pretreated beads to which sperm DNA was not added. More cells were needed for ChIP-seq, and 5 × 10P value cutoff for peak detection was 105. However, the d values (distances between the modes of the Watson and Crick peaks) in the calculated models of AR were <60 bp; therefore, the analysis was done without the MACS model (-nomodel) following the protocol. The parameters were customized to analyze data aligned to the KSHV genome. Because of the smaller size of the KSHV genome, the IgG group was not used as a control for its strong bias; instead, the chromatin input was used as a control in each group. The parameters were set as follows:—bw (bandwidth) 100,—nomodel (without the MACS model),—shiftsize (shift tags by an arbitrary number) 50, and—nolambda . The P value cutoffs at 10−3 and 10−5 were both analyzed. The results were visualized with the integrative genomics viewer (IGV) software [P<10−5).The ChIP-seq data were aligned with the human genome (hg19) and the KSHV genome (HQ404500 plus 35 copies of the TR [U75699.1]) using Bowtie2 ; only onAll the procedures were performed as described in the manual of the luciferase assay system (Promega). The luciferase reporter plasmid was transfected into cells, with and without DHT treatment. Alternatively, the reporter plasmid was co-transfected individually with the plasmid expressing AR-pCDH-CMV-sf3-blast or AR△NTD+DBD-pCDH-CMV-sf3-blast. The total amount of DNA was normalized with an empty vector in transfection. The cells were harvested 36 h after transfection for the luciferase assay. The fold change was calculated by normalizing the internal expression of Renilla luciferase and calibrated by control treatment.Escherichia coli strain BL21 (DE3) expressing GST or GST-fusion proteins was grown in Luria broth medium at 37°C to the exponential phase and cultured for 12 h at 30°C after induction with isopropyl-thiogalactopyranoside. The cells were harvested and resuspended in ice-cold lysis buffer , followed by sonication lysis . The cell lysates were centrifuged at 12,000 rpm to obtain the supernatant, which was combined with Sepharose 4B glutathione resin  for affinity purification following the manufacturer’s protocols. EMSA was conducted as described previously, with minor modifications  A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments.Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Laurie T Krug, PhDAssociate EditorPLOS PathogensBlossom DamaniaSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064***********************The reviewers agreed that the manuscript tackles an interesting and important aspect of KSHV by seeking to define a molecular determinant for gender disparity in KS pathogenesis. That said, there were numerous concerns that will require additional experimentation to support the conclusions of their findings. The reviewers point out the need for additional controls in experiments . They also noted disparate results between assays  and the need for careful interpretation of data using the inducible cell lines. Lastly, statistical analyses were lacking and careful editing are needed to improve clarity throughout the paper.Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: The manuscript by Ding et al. Androgen receptor transactivates KSHV noncoding RNA PAN to promote lytic replication-mediated oncogenesis: a mechanism of sex disparity in KS addresses the molecular mechanism for why there are more cases of KS in males compared to females. The authors focus on the androgen receptor (AR) and demonstrate the ability of AR to upregulate lytic gene expression, especially PAN RNA. While this is an interesting study which attempts to address the large gender disparity in the development of KS, overall some of the data isn’t convincing and in some places the writing is unclear or confusing.Reviewer #2: This report by Mingzhu Ding et al., describes “androgen receptor transactivates KSHV noncoding RNA PAN to promote lytic replication-mediated oncogenesis: a mechanism of sex disparity in KS”. Using anti-AR ChIP analyses, the authors found a region in the PAN promoter binding AR. This binding appears important for KSHV activation and cell migration (infiltration). The story will be more attractive if the authors could extract their data more carefully and have a deletion or mutation control of the identified AR-binding site in the PAN promoter-Luc reporter assays.Reviewer #3: In the manuscript entitled “Androgen receptor transactivates KSHV noncoding RNA PAN to promote lytic replication-mediated oncogenesis: a mechanism of sex disparity in KS” the authors attempts to corelate the relative higher abundance of the KSHV infection in males with the presence of the androgen receptor and the effect of ligand binding to it. The study provides new information about how the androgen receptor is acting as a transcription factor promoting transcription of some of lytic genes including RTA. The study addresses a central question related to KSHV and provided data that has strongly supported their conclusions. There are few minor queries that needs to be addressed.1. The authors showed the expression of the androgen receptor (AR) transcripts in the B-cells and also by western blot. The protein atlas data clearly showed that AR is detected at the mRNA levels in B cells but not at the protein level. How do the authors address this discrepancy? I agree that KSHV infection may increase AR expression but detecting them at the protein level in such high intensity raises my concern. Is the specificity of the antibody carefully validated?2. In Figure 2A, B the authors showed that BJAB and DH75 which are KSHV negative Burkitt’s cell lines express almost undetectable levels of AR, while BC and BCBL both KSHV positive cells have a very strong expression pattern. BCBL in presence of the DHT (AR ligand) has increased expression of the AR. The authors may want to compare cells with isogenic background like BJAB and BJAB-KSHV, or primary B cells infected with KSHV. This will support their conclusions that KSHV is responsible for increased AR expression. The varying cell background can be problematic in that they may have genetically diverse changes which can make interpretation of the results a bit difficult with high confidence.3. The microscopic image of BJAB and BCBL in presence or absence of the DHT shows enhanced expression of the AR in BCBL treated with DHT compared to the BJAB counterpart. However, comparing this data with the western blot  seems a bit contradictory. In the WB the BJAB in presence or absence of the DHT has no detectable expression of the AR but by microscopy they can detect some signal. The fold change of expression in BJAB and BCBL1 in WB and by microscopy is difficult to correlate and needs some explanation. The authors should double check this and provide statistical analysis of these data which will be more convincing.4. The authors mentioned that lentiviral overexpression of the AR itself can induce the lytic reactivation even in the absence of the DHT treatment. The authors explained this finding based on previous published studies where they showed that AR can form complexes with chaperons, HSPs and immunophilins, and gets transported from the cytosol to nucleus. The authors can expand with more emphasis on how this may function in nuclear localization and subsequent viral activation.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: In the introduction, it’s unclear if AR, which is a membrane-associated receptor, is always present in the nucleus to exert functions as a transcription factor or if this function of AR is dependent on additional factors. Is AR and the localization dependent on KSHV infection?Figure 3. The ChIP-seq experiments are difficult to interpret because the IgG controls are not included in figure and according to the materials and methods “the IgG group was not used as a control for its strong bias”. It’s unclear what is the IgG bias and what control was used in it's place. Also it is not stated in the text if the cells used for ChIP-seq are latent or induced iSLK.219 cells. The treatment of the cells is “iSLK.219 cells were pretreated with 10uM DHT or its solvent control MeOH for 48 h”, does this mean that the solvent control was used as the ChIP control instead of the IgG? The authors should include an image of ChIP peaks from the solvent control AR-IP with the DHT AR-IP to determine the difference in AR-IP with and without DHT treatment.Figure 5. The description of the experiments in the text and figure legend was difficult to follow and the interpretation of the results was unclear.Figure 6. In the luciferase assay the authors show the promoter activity from samples that have either the RTA expression plasmid (RTA-pCMV-HA) or the AR expression plasmid (AR-pCDH-sf3). It would be informative to include the promoter activity from samples that have RTA and AR expressed together since that is relevant during reactivation.Figure 7. How do the authors know that the changes in RTA expression  are from the endogenous viral promoter, which they state is regulated in part by AR, and not the doxycycline-inducible RTA that should not be affected by AR?Figure 8. The description in the text concerning the results of the iSLK.219 cell invasion is “PAN RNA is necessary for AR-mediated oncogenesis”, as iSLK are already transformed cell it is inaccurate to state these experiments demonstrate AR-mediated oncogenesis. Yes, the results demonstrate AR contributes to cell invasion but these experiments do not demonstrate oncogenesis.Reviewer #2: The story will be more attractive if the authors could extract their data more carefully and have a deletion or mutation control of the identified AR-binding site in the PAN promoter-Luc reporter assays. Here below are more specifics:1. Introduction: line 84-85, the cited reference #21 is not appropriated and should go with the original publications in 1996 by Sun R., et al. and Zhong W, et al. I don’t know how reliable the data in the cited reference #25 is due to not appropriate controls and should be removed.Fig. 2. Please delete the figure 2C. Since BJAB cells don’t express any AR in the panel 2a and 2b, fig. 2c for BJAB AR staining must be non-specific.Fig. 4. Not sure with the data in figure 4d for RTA AR-binding regions -293-154 and -325- 202 as their binding affinity was ten-fold lower than other two. In addition, the binding ratio in 4d is inconsistent with the gel data in 4e where the RTA1 and 4 had strong binding to AR in the 4d, but less binding in the 4e in agarose gel analysis. Moreover, the figure 3a does not show a ChIP peak for AR binding to these RTA regions and thus conflicts with the data in panels d and e. Why don’t you map the AR binding region (~200 bps) to a smaller region more precisely in the PAN promoter, instead? Please spell out PSA.Fig. 5. It appears the full-length wt AR was never used in the EMSA binding assay, although both truncated forms of AR, in particular the DBD domain-containing short version of AR, displayed a strong binding. However, the data in panel e are problematic and should be deleted due to formation of the protein-probe aggregates that never got into the gel! There is no gel-shift even for the two truncated AR in the absence of any “cold” competitors.Fig. 6. For the panel 6a, ORF57 binds to the 5’ end of PAN RNA , but not to the promoter DNA region. For PAN promoter, Massimelli et al, showed that RTA display only a small activity in the absence of ORF57 (for RNA stability). I am not sure if anyone showed ORF57 specifically and directly binds to the PAN promoter DNA. By including RTA promoter for the luciferase assay, you will need to provide a better evidence why RTA promoter should be included because your data from Fig. 3a conflicts with the data in Fig. 4. Panel 6b Western blot does not have an equal amount of AR expression for every promoter tested in the assays. Panel 6d bar graphs were not aligned well with all individual proteins used in the study. Protein markers should be shown clearly in all WB panels. Why was a pORF57 reporter included in the study? Does ORF57 promoter have AR binding sites? At least, l could not see any ChIP results from this region from Fig. 3. Surprisingly, the ORF57 promoter responded to you AR! A minus AR-binding region in the PAN promoter in the Luc-reporter should be included as a control in Fig. 6.Fig. 7. I don’t think the panels a-c are necessary for your study because it was under Dox induction. Most exciting results are reactivation of KSHV lytic infection by ectopic expression of AR lentiviruses. Where is the data showing the knockdown efficiency of siAR? Was the KD of AR affecting cell viability or growth?Fig. 8. Data in fig. 8b conflict with the data in fig. 8a and the quantitative data in the panel b did not support your observations in the panel a. The same problems for the panels c and d. Data in the panel e are acceptable. The question is what was the knockdown efficiency of siPAN? I am assuming the KD efficiency would be low as the PAN is so abundant.Reviewer #3: There are no major issues**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: Page 2, line 26: “preferentially infects”, while there is a clear evidence demonstrating that KS predominately occurs in males, the line “preferentially infects” suggests that the susceptibility between males and females is also different which has not been documented. The authors may want to reword this sentence for accuracy.Page 2, line 45: “prokaryotic noncoding RNA”, unclear what the authors are attempting to refer to, maybe PAN RNA, but “prokaryotic” is not an accurate word in this sentence.Page 3, line 83-84: “In lytic or abortive lytic-infected cells, the entire viral genomic transcription cascade occurred”, this isn’t completely accurate as during an abortive lytic infection it can fail to proceed at different points during reactivation without the entire transcriptional cascade occurring.Page 7, line 188: Which secondary antibodies were used and what is the manufacturer?Page 8, line 237-238: “Because of the smaller size of the KSHV genome, the IgG group was not used as a control for its strong bias”, if this is the case what was used as a control for non-specific binding?Page 10, line 288: “KSHV DNA was extracted following the manufacturer’s protocol”, what was the kit and manufacturer being used?Page 11, line 306: What is the concentration of doxycycline used for induction?S1 Table: In the section with qRT-PCR primers “qRT-TMPRSS2 F” is listed twice and it is the exact same sequence. It is unclear from the main text in which experiment TMPRSS2 was used in qRT-PCR assays.Reviewer #2: 1. Discussion was not in a good shape and should address various issues from the results.2. It would be better to have someone good in English to proofread the entire manuscript before submission.Reviewer #3: See review**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, Data Requirements:http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here on PLOS Biology: Reproducibility:https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocolsTo enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at  25 Aug 2021AttachmentRevision1-response to reviewers.docxSubmitted filename: Click here for additional data file. 4 Sep 2021Dear Dr. Wang,Thank you very much for submitting your manuscript "Androgen receptor transactivates KSHV noncoding RNA PAN to promote lytic replication-mediated oncogenesis: a mechanism of sex disparity in KS." for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.When you are ready to resubmit, please upload the following:[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments.Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Laurie T Krug, PhDAssociate EditorPLOS PathogensBlossom DamaniaSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064***********************Reviewer Comments :Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #2: No**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #2: No**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.13299-13314, 2007) in addition to miss a non-specific DNA control in their in vitro PAN promoter DNA binding assays with his-tagged ORF57. Thus, the diagram in Fig. 5a should be revised by deletion of the ORF57-binding sites both upstream and downstream of the PAN RNA transcription start site. As an RNA-binding protein, one of major functions of KSHV ORF57 is to stabilize PAN RNA and other target RNAs.Reviewer #2: Authors addressed most of my comments in this revision and the revised manuscript is now in much better shape. However, the author defended that ORF57 binds to PAN promoter DNA by citing a paper back to 2007 by JVI where the authors in JVI publication even realized that “it is unclear whether direct binding of Mta to Rta contributes to our ability to ChIP Mta on the nut-1/PAN promoter in vivo” . If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, Data Requirements:http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: Reproducibility:https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocolsTo enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at References:Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. 6 Sep 2021AttachmentR2-Responses to the reviewer.docxSubmitted filename: Click here for additional data file. 8 Sep 2021Dear Dr. Wang,We are pleased to inform you that your manuscript 'Androgen receptor transactivates KSHV noncoding RNA PAN to promote lytic replication-mediated oncogenesis: a mechanism of sex disparity in KS.' has been provisionally accepted for publication in PLOS Pathogens.Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.Best regards,Laurie T Krug, PhDAssociate EditorPLOS PathogensBlossom DamaniaSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064***********************************************************Reviewer Comments : 16 Sep 2021Dear Dr. Wang,We are delighted to inform you that your manuscript, "Androgen receptor transactivates KSHV noncoding RNA PAN to promote lytic replication-mediated oncogenesis: a mechanism of sex disparity in KS.," has been formally accepted for publication in PLOS Pathogens.We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles  are generated on a different schedule and may not be made available as quickly.Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens. Best regards,Kasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064


Stroke causes great suffering and severe disability worldwide, and rehabilitation following a stroke seeks to restore lost functions. The extent to which stroke patients get access to rehabilitation in Tanzania is not well estimated, and drawing a current picture of the rehabilitation services for these persons is the first step in developing a more effective rehabilitation model in the country.The objective of this study was to establish the characteristics of stroke and its rehabilitation at the Kilimanjaro Christian Medical Centre (KCMC), a consultant referral hospital in northern Tanzania.p-value<0.05 was considered statistically significant.This was a records-based descriptive study in which demographic, clinical, and rehabilitation information of stroke patients admitted to the KCMC between January 2012 and December 2015 was collected and audited. The means, percentages, and proportions were used to summarise the demographic, clinical, and rehabilitation patterns using SPSS version 24.0 software. The chi-squared statistic was used to examine the relationships between categorical variables, and a Of the 17,975 patients admitted to the KCMC during the period of the study, 753 (4.2%) had suffered a stroke, with a mean age of 68.8 ± 16.4 years. The predominant cause of stroke was hypertension, which accounted for 546 (72.5%) patients. A total of 357 (47.4%) patients had various forms of rehabilitation during the admission to hospital. Following a discharge home 240 (31.9%) patients did not return to the hospital for the continuation of rehabilitation.Stroke patients at the KCMC lack access to rehabilitation therapies. Insufficient access to rehabilitation therapies may warrant the need to explore alternative approaches such as tele-rehabilitation technologies in Tanzania. Stroke causes great suffering and severe disability worldwide, and it is the third leading cause of premature disability as measured in disability-adjusted life years ,2. StrokRehabilitation after a stroke usually seeks to restore lost functions to as normal a level as possible. In high-income countries, it is recommended to immediately commence rehabilitation during the admission to hospital followed by patient transfer to intermediate facilities for the continuation of rehabilitation . This opRecently, Tanzania has reported an increase in deaths due to non-communicable diseases . In 2015The purpose of this study was to draw a characteristic picture of stroke and its rehabilitation at the KCMC, which might be the first step in identifying the extent to which stroke patients get access to rehabilitation. This might inform rehabilitation stakeholders regarding the lack of access to rehabilitation and thus lead to the development of more efficient approaches in this regard.This is a records-based descriptive study in which demographic, clinical, and rehabilitation information on stroke patients admitted to the KCMC between January 2012 and December 2015 was collected and audited. The KCMC is a consultant teaching hospital in Moshi, a town in the Kilimanjaro region of northern Tanzania. It is among the top-ranked teaching hospitals in Tanzania with an in-patient capacity of 630, out of which 107 (16.9%) beds are in the medical ward where stroke patients are hospitalised. It has three rehabilitation-related departments set apart. These include physiotherapy, orthopaedic technology, and occupational therapy. The KCMC has no definitive rehabilitation structure. Thus, patients are attended separately as they are referred to the departments by medical practitioners. During the period of this study, there were ten physiotherapists; six occupational therapists; and six orthopaedic technologists. These staffs were available for all inpatients of all sorts of illnesses. Similarly, the same staffs were available for outpatients whose access to rehabilitation is characteristically influenced by socioeconomic and geographical factors, to mention a few.Kilimanjaro is the smallest region in Tanzania with an area of 13,250 square kilometres. It has good roads, which can ease the transport of persons with movement difficulties. However, ambulances are scarce, and public and private transports are widely used to transfer patients, which is not always suitable for patients with movement difficulties. The study was approved by the research ethics committee of the KCMC, Moshi, Tanzania (ref. no. KCMC T.7/61).A data collection sheet was developed to collect demographic, clinical, and rehabilitation information. The name, age, sex, and place of domicile  of all patients admitted to the medical ward between 2012 and 2015 were obtained from the admission registers of the ward. A total of 753 stroke patients’ records were obtained from the register, and the list was submitted to the Health Records Department of the hospital for retrieval of each patient’s relevant clinical information. Demographic information included age, sex, marital status, and place of domicile. Clinical information included the date of stroke, date of admission to hospital, cause of stroke, sub-classification of stroke, outcome , and the date of discharge home. Rehabilitation information included the pattern of access to various forms of rehabilitation therapies, which was based solely on documented evidence of patient attendance and the number of documented contacts between the therapists and the patients.All stroke case records obtained from the period of inquiry were included in the demographics and clinical characteristics aspects of the study. For the rehabilitation part of the study, all stroke records with ‘complete’ information and a diagnosis of stroke were included. Complete information here refers to those case records that had all the information necessary for the audit of rehabilitation of stroke patients. All case records that could not be retrieved, case records from years outside the inquiry period, and duplicate case records that appeared during patients’ transfer from the intensive care unit to the general ward were excluded .Figure 1p-value <0.05 was considered statistically significant. Hospital length of stay was calculated using the admission and discharge dates, and the average length of stay (ALOS) for stroke patients was calculated as the total number of in-patients bed days (HBD) divided by the total number of admissions (discharges and deaths) (DC+D), thus HBD/(DC + D) = ALOS.Prior to the audit of the collected data, age-group categories (years) were constructed according to Tanzania’s age structure  as childp > 0.05). The mean age for the occurrence of stroke for this group of patients was 68.8 ± 16.4 years.A total of 17,975 patients were admitted to the medical ward of the KCMC between January 2012 and December 2015, of whom 753 (4.2%) were stroke patients. Of these, 388 (51.5%) were males and 365 (48.5%) were females giving a male to female ratio of 1:1.06. The distribution between sex and place of domicile was not statistically significant (p > 0.05). The case fatality rate was highest during the first week at 31.7% and reached 39.5% at the end of one month. The ALOS during the admission to hospital was 10.2 days (95% CI 7.1–13.3 days). A total of 463 (61.5%) patients survived the stroke.The overall, in-hospital mortality following admission for stroke was 290 (38.5%), which predominantly occurred in males  compared to females . The majority of deaths (159 (21.1%)) occurred within the first week of the admission to hospital, of whom 70 (9.3%) died within the first 24 hours. The distribution of deaths according to sex, age, or place of domicile was not statistically significant (p > 0.05). A total of 483 (64.1%) records showed that computed tomography (CT) scans had been requested by the physicians, but only 116 (15.4%) had been performed. Of these, 66 (8.8%) were sub-classified as haemorrhagic stroke and 39 (5.2%) were sub-classified as ischaemic stroke. The majority of the patients (479 (63.6%)) were ≥65 years old. Most stroke patients (488 (64.8%)) had left side paralysis.The predominant cause of stroke was hypertension, which accounted for 546 (72.5%) patients. A total of 145 (19.3%) patients had a combination of either HIV or diabetes with hypertension. Causes of stroke between age groups and sex were not statistically significant (p > 0.05).A total of 357 patients had access to various forms of rehabilitation therapies. Less than 50% of them had three or more sessions per week. There was no significant difference between sex, age groups, or place of domicile in terms of being provided with rehabilitation therapies (p < 0.05).A total of 240 patients returned to the KCMC for the continuation of rehabilitation as outpatients. The majority of them (95 (22.1%)) received three rehabilitation sessions per week, making the rehabilitation frequency of 2.3 sessions per week. Age groups and place of domicile significantly influenced the rehabilitation attendance frequency   recommenAbout a quarter of the patients did not get access to rehabilitation therapies. In practice, rehabilitation in Tanzania is not usually accessed without a physician’s referral. This is another reason for physiotherapists’ inability to reach all stroke patients during the admission to hospital. We believe that the physicians delayed referring the patients for rehabilitation because they were seriously ill. Previous authors ,40 have About one-fifth of the patients did not return to the hospital for the continuation of the rehabilitation post-discharge . We could identify no reason for why these patients did not come. However, some patients undoubtedly had died, and others decided perhaps not to continue with the rehabilitation because of reasons reported by previous authors. These include, to mention a few, socioeconomic, stroke-related stigma and limited geographic access to rehabilitation ,35,41,42th week where we could no longer trace the pattern of the attendance. This means that the majority of the patients had substantially accessed rehabilitation for the minimum duration of rehabilitation of 12 weeks while they should have successively access the service for up to 24 weeks. This timeframe is in accordance with evidence-based stroke rehabilitation care described by Bernhardt et al. [Reports show those patients who continued with the rehabilitation progressively reduced their attendance to an average of two sessions per week over three successive weeks. This was followed by attending inconsistently to 12t et al. . We see t et al. ,44 have t et al.  found tht et al.  identifit et al.  and thisIn view of meeting the rehabilitation needs for stroke, the necessary healthcare structure for the rehabilitation of stroke in Tanzania is practically non-existent. We recognise the work done by Giglioni, Bonaventure, Mwinuka and Swai  attemptiThe following are the key findings of this study:Approximately one-third of stroke patients admitted to the hospital do not get access to rehabilitation. Similarly, one-third of patients do not get access to rehabilitation post-discharge.The frequencies of rehabilitation for those who get access to rehabilitation during the admission to the hospital and at post-discharge are lower than recommended.The active rehabilitation duration for stroke patients at the KCMC does not exceed 12 weeks from the time of admission to the hospital.The characteristic picture of stroke in KCMC shows elderly persons (≥65 years) are more affected by stroke (281 (58.7%)) compared with mature working age (55–64 years) group (83 (65.9%)) and prime working age (25–54 yrs) group (99 (69.9%)). Males (388 (51.5%)) are more affected than females (365 (48.5%)) although not statistically significant. The stroke is predominantly caused by hypertension, and the overall in-hospital stroke mortality is 38.5%.Access to rehabilitation for stroke patients at the KCMC is influenced by complex relationships between its tentative rehabilitation structure and patients’ factors. Our findings suggest that interventions that address these modifiable aspects might improve access to rehabilitation. Telerehabilitation technology may improve access to, and outcomes of rehabilitation.The main strength of this study was that it is the first of its kind in Tanzania to report on a characteristic picture of stroke and its rehabilitation. However, it used data from patient files. Therefore, the subjective quality of the documentation of rehabilitation personnel might have had a negative influence on the results. In addition, patients whose incomplete records were excluded from the audit during hospitalisation and those may be considered here as not having access to the rehabilitation during hospitalization, but they may have died before having access to it. Correspondingly, those 117 (32.8%) who did not return to continue with the rehabilitation post-discharge may have died at home, lost interest to rehabilitation follow-up, strangled by variety of reasons as has been reported by previous authors or migrated to other communities making travel to the KCMC unnecessary. Such uncertain data may have additionally biased the findings.


Runners in under-resourced communities in parts of South Africa present with a high prevalence of patellofemoral pain (PFP), which affects their level of participation in sporting activities. Therefore, a specific rehabilitation approach is necessary to manage the PFP-related needs of these runners within their means and reach.To develop a community-based rehabilitation (CBR) implementation framework for PFP amongst runners in under-resourced communities.Our study used the Delphi technique to develop an appropriate rehabilitation implementation framework for PFP in community-based settings. Sport medicine experts, involved in the treatment and rehabilitation of PFP, were recruited to participate. The Delphi process consisted of three rounds to attain consensus amongst the experts on the components and elements that could be contained in a rehabilitation implementation framework for the management of PFP. Experts rated the framework items using a five-point Likert scale.A total of 19 experts participated in our study: 10 were females and 9 were males of whom 13 were aged between 36 and 55 years. Most were local experts (15) with 11–20 years of clinical experience. Four core rehabilitation implementation items were identified through the Delphi process. These were: (1) the establishment of transdisciplinary rehabilitation teams, (2) upskilling of available clinicians, their assistants and trainers, (3) implementation of a CBR programme at low-level or no-cost and (4) referral of cases to secondary or tertiary institutions for further management.Consensus was reached for a comprehensive CBR implementation framework aimed at addressing the specific needs of runners with PFP in under-resourced communities.A further study to test the feasibility of the agreed-upon intervention is recommended. Running is one of the most popular sports that is affordable to many people, especially to those living in poor socio-economic conditions with limited resources. South Africa has an active schedule of running activities that encourages many people to be physically active and provides opportunities for those interested in high-level performance games and races . Unfortunately, running is accompanied by various musculoskeletal injuries including patellofemoral pain (PFP). Patellofemoral pain is considered the most common knee-overuse injury globally  on how rehabilitation programmes for runners with PFP can be implemented in under-resourced communities. In addition, the outcome from studies on the nature of PFP, runners’ needs in under-resourced communities and current rehabilitation strategies were also used .n = 10) with ages ranging between 36 and 55 years (n = 13). The participants’ mean age was 46 years. A total of 15 participants were practising their profession in South Africa and 4 were based in the United Kingdom. The majority (n = 10) had 11–20 years of clinical experience. The mean for participants’ clinical experience was 16 years.Attributes of the Delphi participants are outlined . The stuThe Delphi process yielded four core items of the CBR implementation framework for runners with PFP in under-resourced communities. These were: (1) the establishment of a TDR team (with an average score of 4 out of 4), (2) upskilling of the less skilled (task-shifting) (with an average score of 3 out of 4), (3) implementation of a CBR programme at low-level or no-cost (with an average score of 3 out of 4) and (4) referral of cases to secondary or tertiary institutions for further management (with an average score of 4 out of 4). The framework is outlined in All the 19 participants (100%) agreed that a transdisciplinary rehabilitation team be developed at either secondary or tertiary healthcare centres or at academic institutions that provide outreach to under-resourced communities. The representatives from the athletic federations who serve community clubs will also be included as rehabilitation team members. This rehabilitation team will be responsible for injury screening, planning, monitoring and evaluating the rehabilitation services offered for runners in under-resourced communities.Seventeen (17) out of 19 (90%) participants agreed to the process of identifying and upskilling less-skilled personnel in the local community. As a result of the scarcity of sports rehabilitation services in South African low socio-economic communities, cross-training or the upward skills development of the available community healthcare workers, assistant therapists and club trainers was proposed for the effective implementation of the rehabilitation programmes. The training was proposed to be carried out by the established TDR teams.Eighteen (18) out of 19 (95%) agreed to the implementation of CBR programmes at a low-level or no-cost. Pro Deo services were suggested to be the appropriate method to make services freely available for runners who cannot afford them. These services were proposed to be offered by the upskilled community healthcare workers, assistant therapists and club trainers. Rehabilitation programmes would be structured to encourage clubs to take ownership of their exercise rehabilitation programmes with less involvement of a clinician. Club trainers would have to be fully involved to ensure that the runners comply with their prescribed exercise rehabilitation programmes.All 19 participants (100%) agreed to the referral of cases for further injury management at a secondary and tertiary healthcare facility. The upskilled personnel working with runners in a local community would be responsible for identifying runners whose injuries are not improving with the implementation of the exercise rehabilitation programme and for referring them for further management. Clinicians (members of the established TDR team) would then be expected to further manage the referred cases. A low or no-cost service is still proposed even at this level of intervention.Studies have shown that runners in under-resourced communities have a high prevalence of poorly managed PFP injuries because of inaccessible, unaffordable, unavailable and inadequate rehabilitation services  is synergistic to that proposed in our study. The National DOH  publishePrevention and rehabilitation programmes for PFP consist mostly of exercises that can be taught and delegated  to a less-specialised person in the community to implement, for example, healthcare workers or sports trainers. The exercise rehabilitation programme for PFP may include strengthening exercise of the proximal lower limb muscles including muscles controlling the hip  and knee   system, this framework will be more applicable. The NHI programme is aimed at providing healthcare services that are equally available, accessible, affordable, adequate and appropriate for all South African citizens , which cannot be implemented in the local community, a referral will have to be made for such runners to be attended to at the next level of care, secondary or tertiary institution. We recommend that specialised sports clinics be established at secondary and tertiary levels , where athletes from under-resourced communities may be provided with advanced and specialised services once they have been referred from a primary healthcare level. It is advisable that the government, through the DOH, or the Department of Sports and Recreation  subsidisOur study established a consensus-based CBR implementation framework for athletes in under-resourced communities. The framework proposes the establishment of a TDR team to screen for PFP injuries and plan, implement, monitor and evaluate rehabilitation programmes for runners in under-resourced communities. The rehabilitation team will also be responsible for transferring their knowledge and skills to less-skilled personnel to ensure that the whole community is catered for (a task-shifting approach). Community rehabilitation services are proposed at a low-level or no-cost to make services equally available and affordable for runners. The last step of the proposed framework deals with the referral of cases for further management at secondary or tertiary institutions. For the proposed framework to be rolled out to the targeted communities, a pilot study is recommended to determine its feasibility. The implementation and evaluation of the proposed intervention will then need to be followed.