The search for animal fossils from the Proterozoic eon is hampered by uncertainty about what physical characteristics to expect. Sponges are the most basic known animal type3; it is possible that body fossils of hitherto-undiscovered Proterozoic metazoans might resemble aspect(s) of Phanerozoic fossil sponges. Vermiform microstructure5, a complex petrographic feature in Phanerozoic reefal and microbial carbonates, is now known to be the body fossil of nonspicular keratosan demosponges10. This Article presents petrographically identical vermiform microstructure from approximately 890-million-year-old reefs. The millimetric-to-centimetric vermiform-microstructured organism lived only on, in and immediately beside reefs built by calcifying cyanobacteria (photosynthesizers), and occupied microniches in which these calcimicrobes could not live. If vermiform microstructure is in fact the fossilized tissue of keratose sponges, the material described here would represent the oldest body-fossil evidence of animals known to date, and would provide the first physical evidence that animals emerged before the Neoproterozoic oxygenation event and survived through the glacial episodes of the Cryogenian period.Molecular phylogeny indicates that metazoans  emerged early in the Neoproterozoic era Vermiform microstructure in microbial reefs dating to approximately 890 million years ago resembles the body fossils of Phanerozoic demosponges, and may represent the earliest known physical evidence of animals. Stromatolitologists have struggled for over a century to decipher their microscopic laminae and clots, which are assumed to have been produced or influenced by in vivo and/or post-mortem biogeochemical activity, and to formalize the ‘taxonomy’ of their morphology and microstructure12, and geochemical evidence (biomarkers)13 provides disputed15, indirect evidence for Cryogenian poriferans. The search for definitive physical evidence of pre-Cryogenian metazoans is confounded by uncertainty about what to look for, but preserved physical evidence should be small, subtle and possibly altogether unfamiliar. Given that sponges are the most basic of known animals3, physical evidence of Neoproterozoic sponges could be sought, but effort focused on the characteristics of mineralized sponge skeletons 18 overlooks sponges with only proteinaceous (spongin or keratin20) skeletons. Early metazoan evidence might instead resemble taphonomic  products of sponge soft tissue23 rather than mineralized sponge skeletal components. Although molecular clock data suggest that sponges emerged in the early Neoproterozoic1, the oldest undisputed sponge body fossils are from the Cambrian period15.The existence of metazoans by the Ediacaran period (the last period of the Neoproterozoic) is indicated by bilaterian ‘body’ and trace fossils7 has shown that vermiform microstructure5—an unusual microscopic feature in Phanerozoic reefs and stromatolites that was initially interpreted as being related to algae4 or protozoans25—is instead a keratose sponge body fossil comprising complexly anastomosing cement-filled microtubules enclosed in carbonate microspar. It is produced taphonomically10 in nonspicular keratose demosponges through post-mortem calcification of soft tissue to produce carbonate microspar (automicrite), which surrounds the tough spongin fibres of the ‘skeleton’ of the sponge. Decay of the spongin then produces a network of complexly anastomosing tubular moulds that eventually become passively filled with sparry calcite cement. Although questioned26, the association between vermiform microstructure and sponges has been confirmed in undisputed body fossils of Phanerozoic sponges7. Three-dimensional reconstruction of vermiform microstructure has shown that tubule shape and branching configuration are too consistent and complex to be abiogenic , do not resemble the branching style of other possible organism types 6 and are identical to the spongin meshworks of keratose sponges6. Although the existence of Proterozoic vermiform microstructure has been predicted27, published examples are rare29 and difficult to understand.Recent work22, and produces sponge ‘mummies’22 as well as a range of subtle carbonate sedimentary textures (such as peloid clusters) in living and Phanerozoic fossil sponges22. Taphonomic sediment textures (polymuds) that may be poriferan-related have previously been identified in the reefs that are the subject of this Article23.The calcification of decaying sponge soft tissue has been documented in modern sponges31 Little Dal reefs  36, and developed palaeotopographic relief of up to about 100 m above the surrounding subphotic, level-bottom carbonate-mud seafloor. Reef framework, which is generally not discernible in natural exposures, was documented from slabbed hand samples and thin sections35.This petrographic study presents possible evidence of sponge body fossils in thin sections  from the approximately 890-million-year-old (Ma)da) Fig. . These l36, but stage V records a shallow-water, high-energy environment. The microstructure that forms most of the reefal microbialites  comprises filaments that are about 10 μm in diameter, separated by 10–100-μm masses of marine cement that probably represents the calcified sheath polysaccharide of cyanobacteria36 that was permineralized during microbialite growth. The reef framework, consisting predominantly of this microstructure and its taphonomically degraded equivalents36, defines primary void networks (millimetres to centimetres in size) that are commonly floored with geopetal carbonate mud and lined by isopachous, fibrous marine calcite cement. The relative timing of void-filling by marine cement precipitation versus geopetal sediment accumulation is variable, attesting to the very early timing of marine cement precipitation.The reefs grew in five stages Fig. , each wiVermiform microstructure in samples from stages II, III and V of the Little Dal reefs Fig.  is identVermiform microstructure is present in three microfacies ), representing three palaeoenvironments Figs. , 3. It iIn microfacies i Fig. , 2, vermIn microfacies ii Fig. , vermifoIn rare microfacies iiia Fig. , vermifo37. The compositional and textural homogeneity of the microspar groundmass supports an origin through permineralization of a pre-existing biological substance9, rather than incremental accumulation of detrital sediment or microbial carbonate that passively incorporated complexly anastomosing tubular microfossils. Variable preservation and association with geopetal peloid accumulations are familiar aspects of Phanerozoic sponge taphonomy38. In previous work, detailed comparison of the three-dimensional characteristics of vermiform microstructure with branching cylindrical organism types yielded no convincing alternative to the sponge interpretation6.The shape, size, branching style and polygonal meshworks of the Little Dal vermiform tubules closely resemble both spongin fibre networks of modern keratosan sponges Fig.  and verm40.The preference of Little Dal vermiform microstructure for environments that were not inhabited by photosynthetic calcimicrobes , versus its absence from filamentous calcimicrobial reef-framework components, suggests that (1) illumination may not have been a requirement and (2) the organism may have been unable to compete with reef-building photosynthesizers that grew and/or calcified rapidly. The interlayering of vermiform microstructure with calcimicrobe-free microbialite (microfacies iiib) in the high-energy, well-illuminated reef surfaces of reef stage V supports the interpretation that the vermiform-microstructured organism was not capable of competing with reef-building filamentous cyanobacteria, but instead occupied niches in which the filamentous calcimicrobes did not live owing to (1) poor illumination or (2) high hydrodynamic energy. The occupation of cryptic microniches (shelter and reef framework voids) by sponges , is well known in the Phanerozoic41 except perhaps in the vicinity of photosynthesizing microbial communities; the metabolic requirements of metazoans may have limited early animals to localized, comparatively well-oxygenated (for the time) environments (oxygen ‘oases’). Given the approximately 890-Ma depositional age31, the vermiform-microstructured Little Dal organism may been tolerant of ‘low’ oxygen , which is a characteristic of some modern and fossil sponges42.The obligatory spatial association of vermiform microstructure with reefs built by oxygen-producing cyanobacteria may indirectly support a metazoan interpretation. Prior to the Neoproterozoic oxygenation event, marine dissolved oxygen was probably low45 and (2) early animal life was not catastrophically affected by the Neoproterozoic glacial episodes. If the Little Dal objects are truly sponge body fossils, they are older than the next-youngest undisputed sponge body fossils (Cambrian)15 by approximately 350 million years.If the vermiform-microstructured masses in the Little Dal reefs are accepted as early sponge body fossils, their approximately 890-Ma age would imply that (1) the evolutionary emergence of metazoans was decoupled from the Neoproterozoic oxygenation event46. If the masses of vermiform microstructure in the Little Dal reefs were to be accepted as an early Neoproterozoic expression of sponge tissue preservation, their age and proposed identity would be compatible with (1) evidence that the opisthokont  clade was already established by the time of the Mesoproterozoic–Neoproterozoic transition48, (2) possible evidence of 1-billion-year-old multicellular holozoans49, (3) molecular clock estimates for the emergence of the Porifera in the early Neoproterozoic1 and (4) a revised taxonomy of nonspiculate keratose sponges showing that they are a sister group to other demosponges19. The Little Dal vermiform microstructure is perhaps exactly what should be expected of the earliest metazoan body fossils: preservation through post-mortem calcification of sponge-grade soft tissue in the decaying bodies of small, shapeless, sessile, epibenthic and cryptic animals most closely affiliated with keratose sponges.It would not be surprising to find that the earliest sponges were reef-dwellers; the history of Phanerozoic reefs is rich with reef-building and reef-dwelling spongesField work was done on foot from two-person, backpacking-style camps placed at sites that are accessible only by helicopter. Samples were collected at various times between 1992 and 2018, under all required permits. Recording sample locations using GPS is not possible for most sites owing to the extreme topography of the exposures’ cliffs, pinnacles and canyons, and so sample location was documented using photographs and sketches. Several samples are from a mineral-exploration drill-core stored on-site in the field. Owing to the homogeneous grey weathering of reef surfaces, lithofacies cannot be identified in the field. Instead, hand samples were collected and later slabbed and thin-sectioned. Vermiform microstructure was identified in a small proportion of the samples collected. Repeat visits focused primarily on resampling the rare areas in which vermiform microstructure had been identified.Standard 30-μm-thick thin sections were examined in plane-polarized transmitted light using a Nikon C-Pol binocular microscope fitted with digital camera and Luminera Infinity Analyze software (for lower-magnification images) and an Olympus BX-51 petrographic microscope equipped with Q-Imaging digital capture system (for higher-magnification images).Further information on research design is available in the Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-021-03773-z.Reporting SummaryPeer Review File


Resuscitative endovascular balloon occlusion of the aorta (REBOA) is performed in patients with hemorrhagic shock who develop massive subdiaphragmatic bleeding. This procedure enables rapid and less invasive aortic blockade compared to resuscitative thoracotomy and aortic cross-clamp procedures. However, the REBOA procedure is often blindly performed in the emergency department without fluoroscopy, and the appropriateness of the procedure may be evaluated on computed tomography (CT) after REBOA. Therefore, radiologists should be familiar with the imaging features of REBOA. We present a pictorial review of the radiological findings of REBOA along with a description of the procedure, its complications, and pitfalls. High-energy trauma is defined as open or closed injuries caused by forces . Organs and tissues can sustain extensive damage due to a high amount of kinetic energy, causing severe bleeding. In such time-sensitive and life-threatening situations, urgent endovascular techniques are essential .Resuscitative endovascular balloon occlusion of the aorta (REBOA) is a resuscitation procedure in which a balloon catheter is inserted into the aorta to create a balloon blockade that increases proximal arterial pressure to maintain central organ perfusion while controlling distal subdiaphragmatic hemorrhage . Aortic ™ or Rescue Balloon ER™, Tokai Medical Products Corp., Kasugai, Aichi, Japan) according to the following steps: (1) establish arterial access  and place the sheath, (2) insert the REBOA catheter  over the guidewire, (3) remove the guidewire and insert the stylet into the REBOA catheter to avoid kinking or migration of the balloon catheter because of the arterial pressure during balloon inflation, and 4) inflate the balloon. Ideally, balloon proximal and distal arterial pressure monitoring lines  allow us to determine adequate balloon positioning and inflation [The procedure was performed using a 7-French REBOA device .Zone II is defined as the area between Zones I and III. Placement in Zone II should be contraindicated due to risk of gastrointestinal ischemia.The balloon is positioned in the aorta between the origins of lowest renal artery to the aortic bifurcation. Zone III is indicated in patients with life-threatening pelvic or lower limb hemorrhage .Radiologists must assess the appropriateness of REBOA procedure Figs.  , 4 and dThere are two main types of complications: perfusion-related and procedure-related complications.Perfusion-related complications include distal ischemia–reperfusion injury and exacerbation of proximal bleeding.REBOA can control subdiaphragmatic hemorrhage by decreasing distal arterial pressure; however, it may decrease organ perfusion Fig.  . IschemiREBOA can worsen hemorrhage above the diaphragm  because REBOA-placement-related complications include iatrogenic vessel injuries and inappropriate device insertion .The REBOA procedure can be performed blindly, which may cause loop formation of the guidewire Fig.   or the bUnknown vessel injury or variant artery can potentially lead to device migration during the procedure Fig.  . Device Although REBOA can control subdiaphragmatic hemorrhage, hidden extravasation of the contrast medium may occur Fig.  . The absRenal contusions or renal vascular injury are characterized by decreased enhancement of the renal parenchyma . Renal iThe medical history may be uncertain in some emergency patients. A history of aortic diseases  may cause complications or render REBOA ineffective Fig.  .Fig. 12TThe efficacy of REBOA in the presence of a major venous abdominal injury is unclear. REBOA appears to be effective for central venous injuries in a porcine model ; howeverREBOA plays an important role in patients with severe hemorrhagic shock. Accurate and prompt interpretation of CT findings is essential for treatment and can be a life-saving maneuver.


Minimal-invasive treatment strategies are emerging techniques. Different sources of energy are used to aim for targeted treatment in order to reduce treatment-related complications and morbidity. Imaging plays an important role in targeting and monitoring of treatment approaches preserving parts of the prostatic tissue. Multiparametric magnetic resonance imaging (mpMRI) is widely used during image-guided interventions due to the multiplanar and real-time anatomical imaging while providing an improved treatment accuracy. This review evaluates the available image-guided prostate cancer treatment options using MRI or magnetic resonance imaging/transrectal ultrasound (MRI/TRUS)-fusion guided imaging. The discussed minimal invasive image-guided prostate interventions may be considered as safe and feasible partial gland ablation in patients with (recurrent) prostate cancer. However, most studies focusing on minimally invasive prostate cancer treatments only report early stages of research and subsequent high-level evidence is still needed. Ensuring a safe and appropriate utilization in patients that will benefit the most, and applied by physicians with relevant training, has become the main challenge in minimally invasive prostate cancer treatments.Whole gland prostate cancer treatment,  Despite the radical nature of these elaborate approaches, 40% of the patients undergoing definitive therapy will develop a biochemical recurrence. Furthermore, whole gland treatment also comes with treatment-related complications and side-effects which results in decreased functional outcome measurements.Traditionally, definitive treatment of a prostate tumor includes whole gland therapy, regardless the size or location of the tumor. Whole gland prostate cancer treatments includes radical prostatectomy (RP) or radiation therapy (RT) and is used for treating intermediate- to high-risk prostate cancer. Patients with low-risk prostate cancer mostly opt for an active surveillance policy. The main advantage of these techniques is that preservation of the adjacent structures more likely because of the minimal-invasive approach and consequently treatment-related complications and morbidity could be reduced. Recent studies demonstrate that post-treatment prognosis is predominantly driven by the largest lesion with the highest grade, the so-called “index lesion”. Treatment approaches which preserve parts of the prostatic gland are considered as partial gland ablation and include “hockey stick” ablation, hemi-ablation, and focal ablation. Consequently, imaging plays an important role in detection, localization, targeting and monitoring partial gland prostate cancer treatments.In the last decade, several minimal-invasive treatment strategies are emerging. These treatment methods aim for local destruction of cancerous cells using various sources of energy. It is also used to differentiate between post-treatment changes and potential recurrent or residual disease. As such, secondary treatment can be promptly established. More recently, mpMRI has gained acceptance in image-guided therapeutic settings since it offers real-time anatomical imaging in different planes and therefore improved treatment accuracy. Furthermore, it can provide real-time temperature imaging. However, using MRI during prostate cancer treatment can be time-consuming, expensive and the availability is limited to centers with experienced  radiologists and urologists. Therefore, magnetic resonance imaging/transrectal ultrasound (MRI/TRUS)-fusion guided treatments in which previously obtained mpMRI is cognitively or software-assisted fused with real-time TRUS images are more and more performed. Using MRI/TRUS fusion has the benefit of relatively low-costs because no MRI-compatible equipment and extra MR-scanner time are needed and it is readily available compared to the use of MRI. However, accurate alignment of the MR images is essential for a successful image registration and some MR reading experience is needed for accurate interpretation of the images.Multiparametric magnetic resonance imaging (mpMRI) is preferred in detecting and staging prostate cancer due to excellent soft tissue contrast and multiplanar anatomical imaging.The aim of this review is to provide an overview of the available techniques for image-guided prostate interventions using MRI and MRI/TRUS-fusion imaging. i.e. repeat FLA or radical treatment) remain a viable option after initial treatment with FLA.Prostate cancer treatment using focal laser ablation (FLA) or Laser Interstitial Thermo Therapy (LITT) is based on thermal ablation. An optical laser fiber is applied within the cancerous tissue by either a transrectal or transperineal approach. Energy provided by the laser raises the temperature of the targeted tissue above 60°C, this results in direct focused cell death. The number of ablations that is needed to cover the targeted area depends on the volume of the intended lesion and the type of laser fiber that is used. The total procedure time depends on the number of ablations that is needed, with an average of only a few minutes per fiber position. This results in short procedure times and a fast post-treatment recovery. Most importantly, secondary interventions  become available for FLA.Transperineal FLA is mostly performed under general or spinal anesthesia, where patients are placed in a lithotomy position. The laser fibers are usually placed with the help of a transperineal template grid. Transrectal FLA is commonly performed under local anesthesia or under sedation as an outpatient procedure, where patients are placed in prone position.  but can also be used in a salvage setting. A recent study by Walser et al demonstrated a freedom of retreatment rate of 83% after a 1 year follow-up in a group of 120 men with low- to intermediate-risk disease that underwent transrectal FLA with no significant changes in quality of life or sexual and urinary function. Despite recent studies demonstrating large numbers of low- and intermediate-risk disease, the long-term follow-up is still lacking, and therefore patient selection and eligibility criteria need to be carefully evaluated based on which patients will benefit the most.The use of FLA appears to be a safe and feasible option for patients with MRI-visible and biopsy-proven localized low- to intermediate-risk prostate cancer who are eligible for both active surveillance (AS) or radical treatment,Cryoablation is based on the administration of alternating freeze and thaw cycles which will induce cell death and subsequently destruction of the targeted tissue. Tissue injury mainly occurs by cellular dehydration due to the decreased temperature (−40°C) of extracellular water and results in an osmotic gradient followed by coagulative necrosis, thrombosis and tissue ischemia. The process is enhanced by intracellular formation of ice crystals, causing a complete cell disruption.® by Galil Medical, Inc and Endocare Cryocare SL® by HealthTronics, Inc.). Monitoring the ice-ball formation using TRUS is limited due to acoustic shadowing. Consequently, the completed coverage of the ice-ball remains unknown, which may lead to detrimental freezing of the adjacent structures.Cryoablation is performed under general or spinal anesthesia with the patient placed in lithotomy position. Traditionally, the cryoprobes are transperineally inserted using TRUS guidance as it allows real-time feedback on probe positions as well as ice-ball formation. The number of cryoprobes needed depends on the size and shape of the intended ablation zone as well as the type of probe that is used. Nowadays, two prostate cryotherapy systems are used  is used to heat (>60°C) and destroy the targeted prostatic tissue. The focused ultrasound pulses induce immediate and irreversible tumor necrosis and cause cavitation with sharply delineated margins. A HIFU procedure is performed under local or general anesthesia while using real-time TRUS, MRI or MRI/TRUS-fusion guidance for positioning and monitoring of the transrectally inserted probe. The damage that is caused by using HIFU is limited to the heated tissue, therefore it aims to lower the risk of treatment related side-effects while preserving quality of life and potency. This also allows secondary treatment using HIFU or additional radical salvage therapy if necessary. Because the penetration depth of the HIFU pulses is limited, HIFU is considered less suitable for large prostates (>40 cc) or for anterior tumors. Sometimes, a transurethral resection of the prostate (TURP) is performed before HIFU to diminish the prostate volume and to make the prostate eligible for HIFU-treatment.High-intensity focused ultrasound (HIFU) is a rapidly growing minimal non-invasive partial gland treatment option. Energy from a high-frequency ultrasound transducer  for patients with clinically significant nonmetastatic disease. The oncological outcomes demonstrate an improvement over time, indicating that selection criteria and expertise of the physician are pivotal in the application of focal HIFU.To date, HIFU is mostly used as focal prostate cancer treatment in both ® technology. It combines MR-based treatment planning and real-time monitoring with thermometry while ablating prostate tissue using transurethral thermal ultrasound. By inducing thermal coagulation of the prostatic tissue, TULSA can deliver accurate and precise whole gland or partial gland ablation. Treatment with TULSA differs from HIFU by delivering a continuous targeted ultrasound beam directly from the urethra, rather than various transrectal focused ultrasound spots. The TULSA-PRO® technology includes a urethral applicator with a linear array of 10 ultrasound transducers that provides a beam of focused energy into the prostatic tissue in order to achieve a temperature of >55°C in the targeted tissue. The urethral applicator and endorectal cooling device facilitate periurethral and rectal preservation by active water cooling. The TULSA procedure can be performed as outpatient procedure and requires general or spinal anesthesia. MRI-guided transurethral ultrasound ablation (TULSA) is a novel procedure using the TULSA-PRO describes whole gland MRI-guided transurethral ultrasound ablation in 115 men with predominately intermediate-risk prostate cancer. While sparing the urethra and apical sphincter, the MRI-guided TULSA demonstrated PSA reduction combined with a low morbidity-rate. Targeted sampling of the treatment area during the 1-year follow-up demonstrates no residual or recurrent tumor in almost 80% of the patients. TULSA also benefits the preservation of the adjacent tissues with a relatively low risk of functional problems. None of the patients had a rectal injury, 96% returned to baseline urinary continence and 75% of potent males maintained or returned to erections sufficient for penetration. Recently, Attinen et al showed promising early-stage oncological control and low toxicity in patients undergoing TULSA as salvage treatment for local recurrence after primary radiation. 90% of the patients were free of disease in the targeted ablation area one year after treatment.A large prospective multicenter study by Klotz et alDespite the promising results, further studies on long-term oncological outcome and treatment-related side-effects are still warranted. Moreover, studies investigating the potential of focal TULSA are still ongoing and the results have not yet been published. Consequently, PDT has shown to initiate selective cytotoxicity towards malignant cells and subsequently leads to malignant cell death. The commonly used photosensitizers differ in form of administration , wavelength required for activation (405–763 nm) and targeted tissue (tissue-based or vascular acting). However, photosensitivity and phototoxicity are important factors associated with PDT and need to be considered as main limitations. The intracellular accumulation of the photosensitizing drug inside of cells might lead to increased susceptibility to prolonged phototoxicity, resulting in an extreme sensitivity to ultraviolet (UV)-rays from the sun and other light sources.Photodynamic therapy (PDT) is a minimally invasive prostate cancer treatment. Its principle is based on three main components: a photosensitizer, light, and tissue oxygen. After local administration in the prostate, a photosensitizing drug is activated by low-power laser light delivered by optical fibers. The optical fibers are placed under TRUS or MRI/TRUS guidance using a needle and a perineal template as guidance. Transfer of energy from the activated photosensitizing drug to biological substrates or molecular oxygen, generates reactive oxygen species which induces cell death by apoptosis or necrosis. Compared to active surveillance, PDT demonstrated to be a safe and an effective tissue-preserving approach for low-risk localized prostate cancer. In these cases, PDT may even defer or avoid radical therapy. Nowadays, studies are mainly focusing on applying PDT as treatment of localized advanced prostate cancer and isolated metastases. Treatment of advanced prostate cancer using PDT has the potential to establish annihilation of the malignant tissue and reduce damage to adjacent structures. The oncological efficacy of PDT on androgen-refractory prostate cancer is especially important for prostate tumors or patients resistant to hormonal therapy. The use of PDT also appears suitable for organ confined recurrent prostate cancer after radiation therapy, destroying essentially all glandular tissue within the prostate under precise light dosimetry with only a few complications. However, the chronic and cumulative toxicity that is associated with radiation therapy does not occur during PDT. More importantly, PDT can be applied to already irradiated prostatic tissue.Photodynamic therapy has the potential to be very targeted and provides a single session treatment, nevertheless it can also be used in primary (whole gland) or salvage settings. Prostate cancer treatment using RFA can be an effective and safe option for patients with clinically localized prostate cancer as well as patients with non-metastatic recurrent disease. It is used as primary or salvage therapy for cancers that are not eligible for surgical removal and a common treatment option in case of therapy-resistant tumors.Radiofrequency ablation (RFA) or radiofrequency interstitial tumor ablation (RITA) is an innovative targeted treatment that precisely delivers low-dose radiofrequency waves directly to cancerous tissue. The targeted tissue is irreversibly destroyed by the established coagulative necrosis and atrophy. Recently, Orczyk et al reported on focal MRI/TRUS fusion-guided bipolar radiofrequency ablation for clinically significant prostate cancer visible on mpMRI in a group of 20 patients. This bipolar “Encage” system  uses a novel asymmetrical coil which creates a precise uniform zone of coagulative necrosis. 6 months after treatment, 80% (n = 16) was free of clinically significant prostate cancer on targeted transperineal biopsy. Absence of erectile dysfunction was seen in 91.7% of the patients with no erectile dysfunction at baseline. The return to baseline mainly occurred during the first 6 weeks after treatment. Scores for intercourse satisfaction, sexual desire, overall sexual satisfaction and orgasmic function did not demonstrate any changes. In 89% (16/18) of the males with no urinary incontinence at baseline the absence of urinary incontinence remained after treatment. Furthermore, they reported no change in lower urinary tract symptoms, bowel habit, general health and prostate related quality of life. The use of focal RFA is a feasible and safe treatment option for patients with localized prostate cancer. However, further research with extended follow-up is needed to evaluate oncological efficacy and treatment related side-effects.Traditionally, RFA is performed under general or spinal anesthesia, where patients are placed in a gynecological position for a transperineally approach. A transurethral catheter is needed to improve ultrasonographic visualization. The needles are placed under TRUS or MRI/TRUS fusion guidance while using a transperineal template grid. A generator  provides monopolar or bipolar radiofrequency energy up to 50 W at a frequency of 480 kHz. The electrical pulses produce irreversible cell membrane permeabilization which causes apoptosis of the cells. Therefore, it only affects the cell membrane while preserving the surrounding tissues and the extracellular matrix.Irreversible electroporation (IRE) is a relatively novel non-thermal ablation technique where micro- to millisecond electrical pulses travel between transperineally inserted electrodes.IRE procedure is performed under general anesthesia and deep muscle paralysis using TRUS or MRI/TRUS–fusion technique for electrode placement. Patients are positioned in lithotomy position and a transurethral urinary catheter needs to be placed. The positioning and guidance of the electrodes requires a transperineal template grid. Most IRE procedures are performed by using a widely available IRE generator with planning software . The average procedure time using IRE is approximately 1 h. Blazevski et al demonstrated a disease-free survival rate of 97.3% in 123 patients with localized clinically significant prostate cancer. They also defined that the ideal patient should have a biopsy proven intermediate-risk localized prostate cancer with a unifocal lesion on mpMRI. A study by Collettini et al showed a similar cancer-free survival rate of 82% 6 months after IRE. 1-year follow-up data demonstrated a significant reduction in the serum PSA-levels, a stable urogenital function, and a leak- and pad-free continence rate of 96.3%. Remarkably, Scheltema et al experienced more early oncological failure after IRE in a study that compared IRE (n = 50) to robot-assisted radical prostatectomy (RARP) (n = 50). However, they demonstrated superior preservation of pad-free continence (UC) and erections sufficient for intercourse (ESI) in the group that underwent IRE. 12 months after treatment, urinary symptoms had been reduced for both groups, although the patients undergoing IRE initially had more urinary symptoms directly after treatment. Additionally, males with a poor baseline functioning are more likely to develop erectile dysfunction after IRE. Despite the promising oncological outcomes, long-term follow-up data of oncological efficacy and treatment-related complications and side-effects is currently not available.Multiple Phase I–II studies have shown that IRE is a safe and feasible focal treatment option with a low morbidity rate in both primary and salvage diseases.The principle of brachytherapy  is based on radioactive seeds that are implanted within the prostatic tissue under image guidance. Brachytherapy can be distinguished by high-dose rate therapy (HDR) or low-dose rate therapy (LDR). High-dose rate brachytherapy is delivered in a brief treatment session where radioactive pellets are temporarily inserted. This results in accurate dosimetry as it allows modulation of the source dwell time and implant position. Alternatively, LDR brachytherapy allows definitive implantation of tiny radioactive titanium seeds and is more commonly used as it is most suitable for low-risk prostate cancer in low volume prostate tissue. i.e. EBRT due to a specific distribution of high dose radiation. However, brachytherapy may also be combined with EBRT in order to improve prostate cancer treatment. Partial gland therapy using brachytherapy is mainly performed transperineally under either MRI or MRI/TRUS-fusion guidance while using a biopsy template. During treatment under local, spinal or general anesthesia, the patient is placed in lithotomy position and a transurethral foley catheter is inserted. In case of cognitive or rigid fusion systems, the mobility and deformation of the prostate influence the precise targeted area, Therefore, the implantation of brachy seeds using elastic fusion registration is evaluated. This ultra-focal HDR brachytherapy, uses a single ancillary ultrasound visible marker that is transrectally inserted within or close to the targeted region prior to routine brachytherapy. A recent study by Graff et al researched precision delivery of therapeutic radiation doses to small tumor lesions on mpMRI.Brachytherapy can be used as whole gland or partial gland treatment of a prostate tumor with fewer side-effects than,  Most importantly, targeted lesions in the base of the prostate are more likely correlated with urinary symptoms compared to lesions located in the apex during early follow-up after focal brachytherapy. Focal brachytherapy is an emerging prostate cancer treatment which still requires further evaluation to conquer imprecision in the definition of the target  and to ensure collaboration between the imaging modalities required for targeted treatment.Mid-term results of treatment with MRI-guided brachytherapy indicate that it is feasible to treat tumors, while limiting toxicity and preserving quality of life. External beam radiotherapy (EBRT) and its hypofractionation equivalent, stereotactic body radiation therapy (SBRT), are currently widely used in approximately one-third of patients with localized (cT1c-T3N0M0) prostate cancer. However, safe delivery of the intended fraction for tumor tissue in the abdominal and pelvic region is limited due to inter- and intrafractional organ movement. The use of MR-guided radiotherapy (MRgRT) as stereotactic therapy allows for greater accuracy of fraction delivery using better soft-tissue contrast, real-time MR imaging for direct tracking and daily online adaptive planning software. This will improve cancer outcome while reducing the risk of treatment-related toxicity and additional radiation exposure. To date, two commercially available systems are used: Elekta Unity  using 1.5 Tesla, and Viewray MRIdian MR Linacs  using a 0.35 Tesla MRI.Radiation therapy (RT) is generally used as radical treatment for localized prostate cancer in males not eligible for surgery. The average MRgRT dose delivery takes approximately 45 min and has dosimetry benefits over other forms of radiotherapy. A Phase-II study (n = 101) reported a low incidence of early gastrointestinal and genitourinary toxicity using both clinician- and patient-reported outcome measurements in patients with localized prostate cancer undergoing MRgRT. Follow-up data of one study provide extended toxicity information with symptoms no longer present 12 months after treatment. However, studies in a large cohort of patients and long-term follow-up regarding oncologic outcomes are lacking.Magnetic resonance-guided radiotherapy has shown promising results as a safe and tolerable prostate cancer treatment.This review provides an overview of image-guided prostate cancer treatment options while using MR-, TRUS- or MRI/TRUS fusion imaging. Minimal invasive, image-guided prostate interventions may be considered as a viable partial gland ablation option compared with the current, standard treatment options in patients with organ confined low- to intermediate-risk prostate cancer (recurrence). Correct utilization of a treatment in patients that will benefit the most while ensuring a safe and responsible application by physicians has become the main challenge. Majority of the studies focusing on minimally invasive prostate cancer treatments only report early stages of research and high-level evidence is still lacking. Despite the promising results and emerging evidence, definite proof of oncological efficacy compared to radical treatment using randomized controlled trials is required.


This retrospective cohort study aimed to investigate the effects of neonatal oxygen care and retinopathy of prematurity (ROP) treatment on ROP-related ocular and neurological prognoses. We included premature infants treated for ROP at a tertiary referral center between January 2006 and December 2019. Demographic and clinical data were collected from electronic medical records. Odds ratios (ORs) of oxygen care- and ROP treatment-related factors were calculated for ocular and neurological comorbidities 3 years after ROP treatment, after adjusting for potential confounders. ROP requiring treatment was detected in 171 eyes (88 infants). Laser treatment for ROP  and duration of invasive ventilation  were associated with an increase in ocular comorbidities, along with a history of neonatal seizure  and chorioamnionitis . No oxygen care- or ROP treatment-related factors showed significant odds for neurological comorbidities. Shorter duration of invasive oxygen supply during neonatal care (less than 49 days) and anti-vascular endothelial growth factor injection as the primary treatment for ROP are less likely to cause ocular comorbidities. No association was identified between ROP treatment modalities and the risk of neurological comorbidities. The primary treatment of ROP is gradually changing from laser photocoagulation 2 to anti-vascular endothelial growth factor (VEGF) injection3, although neurodevelopmental and systemic outcomes are still under investigation4. In recent studies, ROP treatment-related ocular and systemic outcomes of these infants, including neurodevelopmental impairments, were comparable between different ROP treatment modalities8. However, the effects of changes in perinatal risk and neonatal oxygen care have not been comprehensively investigated in tandem with the change in primary treatment of ROP.Retinopathy of prematurity (ROP), a leading cause of childhood blindness, is a proliferative retinal vascular disease exclusive to premature infants9. Concurrently, the rate of multiple births from artificial/assisted fertilization has increased10 and the rate of age-dependent pregnancy complications is also increasing11. Consequently, the pattern of perinatal risk factors has changed over time13. Changes in perinatal factors, including neonatal care in prematurity, may affect ophthalmic and systemic outcomes of these infants, not limited by ROP treatment factors.The survival rate among extremely premature infants (less than 28 weeks gestation) is increasing14. Presently, ventilator-related strategies aim for lower oxygen targets and higher arterial carbon dioxide levels16. A correlation was found between prematurity, oxygen levels, and ROP risk19. However, little is known about the impact of changing trends in oxygen therapy on ROP-related prognoses.In neonatal care, the optimal approach to oxygen management remains unclear. Lower oxygen saturation levels increase the risk of mortality, while higher targets are associated with increased rate of oxidative stressTo determine which factor related to neonatal oxygen care and ROP treatment, is significant to ROP-related ocular and neurological outcomes, we retrospectively investigated demographic and clinical data in infants treated for ROP over a 14-year period and evaluated possible associations.A total of 786 premature infants were screened for ROP, and type 1 ROP requiring treatment was diagnosed in 118 infants, with an overall incidence of 15%. Thirty infants were excluded due to ineligibility (17 for incomplete follow-up and 13 for missing data) and a total of 171 eyes (88 infants) were included in the analyses. The mean follow-up period was 46.6 ± 10.3 months.Baseline clinical characteristics of ROP infants are provided in Table Perinatal risk factors detected prior to ROP treatment were described in Table Table Supplementary Table P < 0.001).Analysis of the optimal cut-off for the duration of invasive ventilation, using criterion based on Youden’s index, identified an optical cut-off value of 49 days Fig. . This thSupplementary Table Higher birthweight  showed decreased odds of neurological comorbidities. Longer duration of incubator-based care , a history of intraventricular hemorrhage , neonatal seizure , and hydrocephalus requiring shunt placement  had significantly increased odds of neurological comorbidities. Neither low-risk ROP nor APROP appeared to be associated with neurological comorbidities.This study shows that less invasive oxygen therapy may result in more favorable ROP-related ocular outcomes in conjunction with primary anti-VEGF therapy for ROP. In addition, a history of chorioamnionitis or neonatal seizure was associated with ocular comorbidities. The choice of ROP treatment (anti-VEGF injection or laser photocoagulation) and anti-VEGF agent (bevacizumab or ranibizumab) appears to have no significant effect on ocular or neurological outcomes, including neurodevelopment.Among the factors related to neonatal care, the duration of invasive ventilation was associated with an increased risk of long-term ocular comorbidities, whereas ROP severity at the time of treatment did not show a significant association. This result suggests that prolonged invasive ventilation may adversely affect the post-treatment course of ROP. Additionally, the length of incubator care was associated with more common neurological comorbidities. For optimal treatment outcomes in premature infants, our study results indicate that comprehensive multidisciplinary management involving neonatologists, pediatric neurologists, and ophthalmologists is necessary.Overall, infants who recently received ROP treatment were more frequently associated with artificial/assisted pregnancies. This finding is likely accounted for by increasing maternal age, advances in assisted fertilization techniques, and an increasing frequency of artificial/assisted fertilization. Among maternal- and birth-related factors, the presence of maternal chorioamnionitis was a strong independent risk factor for ROP-associated ocular comorbidities. With improved survival rates of extremely premature neonates, the risk of systemic comorbidities has also increased. Importantly, a history of neonatal seizure was associated with an increase of ocular comorbidities. Therefore, infants with a history of maternal chorioamnionitis or neonatal seizure might require careful screening for ophthalmic comorbidities. As expected, a history of neonatal seizure or brain shunt operation for hydrocephalus was a risk factor for neurological comorbidities, along with a lower birthweight.21. Prolonged antenatal action of proinflammatory cytokines, major mediator of chorioamnionitis, have been shown to cause delay in retinal vasculature growth, thinning of the choroid, and long-term morphological alterations of the retina22. Our findings provide real-world evidence that maternal chorioamionitis is a risk factor for ocular comorbidities. While neonatal seizures are often associated with retinal hemorrhages, they have not been shown to actually cause retinal hemorrhages24. However, acute symptomatic seizures can occur due to hypoxic-ischemic encephalopathy, acquired structural brain lesions, metabolic disturbances, or systemic infections, which may cause vascular instability and prolonged inflammation that may affect retinal development25.Chorioamnionitis has been previously associated with retinal and subretinal vasculopathy. In addition to antenatal and postnatal inflammation, circulatory instability and oxygen satuation fluctuations following inflammation may affect retinal perfusion26. However, this is likely to be the result of differences in the age and follow-up between the two groups, warranting caution when interpreting the results. The primary method of ROP treatment (whether laser or injection) was not associated with long-term neurologic prognosis, including neurodevelopmental status assessed by the Denver II Developmental Screening Test. The effects of the anti-VEGF agent type (bevacizumab or ranibizumab) were not significant at risk for neurological comorbidity . This is noteworthy because the potential risk for neurodevelopmental delay caused by systemic absorption of intravitreal anti-VEGF is the main cause of concern regarding widespread use in premature infants. When accounting for comprehensive pediatric evaluations and in depth review of medical records, no increased risk stemming from the use of anti-VEGF therapy was shown over laser treatment. Further research is required to confirm the systemic effects of anti-VEGF agents.In this study, anti-VEGF injection, compared to laser photocoagulation, was associated with a lower incidence of ocular comorbidities. This difference was most apparent in the number of cases undergoing strabismus surgery that may indicate errors in neurodevelopment as suggested from the pivotal ETROP trialsIn the present study, we noted a significant proportion of infants of GA > 32 weeks treated for ROP in the recent period, which is atypical for infants within this age-group to progress to treatment-requiring ROP. There were no periodic changes in the PMA at treatment, suggesting a faster ROP progression rate among infants of higher GA and indicating that careful observation of neonates may be warranted during the critical PMA window (37–39 weeks).27.Recently, interventions appeared to be delivered at an earlier disease stage (ROP classification of zone 2 stage 2 with plus disease), although this difference was not significant. This finding might be due to the simplification of the treatment process from laser photocoagulation to anti-VEGF injection, which might have allowed ophthalmologists greater ease in recommending interventions to eligible neonates with ROP. Furthermore, higher retreatment rates were observed among eyes treated with anti-VEGF. This may be explained by the pharmacokinetics of these anti-VEGF agents and the fact that a higher proportion of eyes were treated with injection for zone 1 disease and APROPThe limitations of this study include its retrospective nature, lack of a control group, and a homogenous sample, which involved a single ethnic group from a well-developed country with easy access to healthcare. Additionally, we could not account for target levels of oxygen therapy due to the retrospective nature of this study. Instead, the application rate and duration of each oxygen therapy modality were used as variables to consider. Because this study was not a randomized controlled trial, we cannot exclude the possibility of confounding bias caused by heterogeneous and time-varying underlying systemic conditions. Therefore, our presentation of possible associations should be considered with caution, and not be interpreted as a cause-and-effect relationship. Furthermore, although variance inflation factor of all independent variables has been evaluated, collinearity between variables may limit our interpretations.However, this study was conducted on a relatively large number of treated ROP eyes (N = 171) over a 14-year period at a large tertiary medical center with an average follow-up period greater than 3 years. Considering the paucity of treated ROP cases that considered the neonatal management and the difficulty of regular follow-up with infants, our study results do have clinical importance.Taking into account the contextual changes in perinatal risk and neonatal care, we have identified some important modifiable factors that may affect the long-term ocular and neurological prognosis of premature infants treated for ROP. In conclusion, shorter application of invasive ventilation during neonatal intensive care unit (NICU) care (less than 49 days) and ROP treatment via anti-VEGF injection have emerged as controllable factors that might improve ophthalmic prognosis among neonates treated for ROP. We were unable to confirm previous concerns regarding a potentially harmful association between anti-VEGF injection or injection drugs and neurological outcomes, in this clinical context.This retrospective study was conducted at Gangnam Severance Hospital, a tertiary referral-based hospital affiliated with Yonsei University College of Medicine. At this hospital, neonates receive care from a multidisciplinary team, including neonatologists, ophthalmologists, anesthesiologists, and other pediatric specialists. Infants requiring ROP treatment were treated at NICUs. This study was based on data extracted from electronic medical records.28; treatment was indicated for infants diagnosed with type 1 ROP, according to the same criteria as used in a previous study2. We further categorized a low-risk ROP type as zone 2 stage 2 ROP with plus disease to determine any possible subtle differences in disease progression and outcomes.Consecutive infants who were treated for type 1 ROP between January 2006 and December 2019 and followed up at least 3 years were included. Neonates were screened for ROP if they met one or more of the following criteria: GA < 32 weeks, birthweight < 1500 g, or unstable clinical course determined by the primary neonatologist based on the applicable protocol at our institution. All ophthalmic examinations were performed by qualified pediatric retinal specialists using the 2005 International Classification of Retinopathy of PrematurityFirst-line treatment for ROP was chosen after discussion with the infants’ guardians and was performed by pediatric retinal specialists. Although varied due to the specialist’s preference, treatment period, and disease severity, the overall treatment decision could be broadly categorized according to the widening availability of different modalities. Prior to the introduction of anti-VEGF injections for ROP treatment, laser photocoagulation was the treatment of choice. The reporting of reliable safety data regarding anti-VEGF use in neonatal infants has made the injection the primary choice for ROP treatment, especially in less severe cases. In addition, the primary anti-VEGF choice switched from bevacizumab  to ranibizumab , after ranibizumab has been shown to have faster systemic clearance and is theoretically safer for use in neonates.Laser photocoagulation was performed under general anesthesia, using an indirect diode laser system with a 30-diopter (D) hand-held aspheric lens following scleral depression, with power settings adjusted to achieve a pale white burn of the retina. The laser was applied to the avascular area. For intravitreal anti-VEGF injection, either bevacizumab or ranibizumab was injected into the diseased eye under sedation and topical anesthesia. The use of these off-label anti-VEGF agents for treating ROP was approved by the Institutional Review Board. Infants were re-examined the following day and every week thereafter to monitor disease progression. Reappearance of plus disease or progression of ROP stage despite adequate treatment were defined as recurrence. As required, experienced retinal specialists performed additional procedures such as injection, laser, or surgery, including vitrectomy or scleral buckling.Clinical characteristics of interest included GA, birthweight, ROP-related factors , and other perinatal factors such as maternal- or birth-related complications, neonatal care , and systemic comorbidities detected prior to ROP treatment. Invasive ventilation was defined as infants with endotracheal intubation or mechanical ventilation, non-invasive ventilation as infants with face or nasal masks, and oxygen supplementation as infants with oxygen hoods. Target oxygen saturation levels are between 90 and 95% for preterm neonates, while mature infants (GA > 34 weeks) with decreased risk for ROP have a higher target of 97%. The use and duration of each category oxygen were individually tailed in the NICU by trained healthcare professionals; this information is saved to the electronic health records of these infants, and we documented these factors for assessment in this study.Adverse systemic outcomes were determined through a complete review of patients’ medical records, which included comprehensive examination of follow-up data provided by pediatric specialists; any diagnoses added by the primary pediatrician after ROP treatment were included as systemic comorbidities. Neurological examinations were performed immediately after discharge from the NICU and the Denver II Developmental Screening Test was routinely performed at regular follow-up intervals in the pediatrics department, and those who failed in two or more items of the test were suspected of having neurodevelopmental delay. Ocular comorbidities included any ROP-related ophthalmologic abnormalities  that can result in poor visual prognosis detected during ophthalmologic follow-up.The primary outcomes were possible associations between methods of oxygen care and ROP treatment with ROP-related ocular and neurological comorbidities, after adjusting for potential confounders.P < 0.10 in the univariable logistic regression analysis were included in a multivariable logistic regression model. Optimal cut-off thresholds were analyzed using criterion based on Youden’s index, which maximizes the sum of sensitivity and specificity and also maximizes concordance . All data are presented as means (standard deviations) for continuous variables or numbers with percentages for categorical variables. P < 0.05 were considered statistically significant.Sample size of 84 cases was calculated with type 1 error probability of 0.05 and power of 80%. We performed statistical analyses using the Statistical Package for the Social Sciences software . Listwise deletion was used method in handling missing data. The Kolmogorov–Smirnov test was used to analyze the distribution of the data. Chi-squared tests for categorical variables were used to compare the groups. Logistic regression analysis  was performed to assess risk factors for ocular and neurological outcomes. Factors with Ethical approval was obtained from the Institutional Review Board of Gangnam Severance Hospital (No. 3-2019-0332). Informed consent for treatment was obtained from the patients’ parents or guardians, and the need for consent to be involved in this study was waived due to the retrospective nature of the study. The study adhered to the tenets of the Declaration of Helsinki.Supplementary Tables.


Lipodystrophic mice are protected from cartilage damage following joint injury. This protection can be reversed by the implantation of a small adipose tissue graft. The purpose of this study was to evaluate the relationship between the gut microbiota and knee cartilage damage while controlling for adiposity, high fat diet, and joint injury using lipodystrophic (LD) mice. LD and littermate control (WT) mice were fed a high fat diet, chow diet, or were rescued with fat implantation, then challenged with destabilization of the medial meniscus surgery to induce osteoarthritis (OA). 16S rRNA sequencing was conducted on feces. MaAslin2 was used to determine associations between taxonomic relative abundance and OA severity. While serum LPS levels between groups were similar, synovial fluid LPS levels were increased in both limbs of HFD WT mice compared to all groups, except for fat transplanted animals. The Bacteroidetes:Firmicutes ratio of the gut microbiota was significantly reduced in HFD and OA-rescued animals when compared to chow. Nine novel significant associations were found between gut microbiota taxa and OA severity. These findings suggest the presence of causal relationships the gut microbiome and cartilage health, independent of diet or adiposity, providing potential therapeutic targets through manipulation of the microbiome. Specifically, taxonomic changes in gut microbiota composition have been associated with OA onset and progression, especially with obesity3, a primary risk factor for OA3. While reports on the relationships between gut microbiota and cartilage damage to date have been largely correlative, fecal transplant from metabolically compromised human donors accelerates OA in germ-free mice challenged with a knee injury, suggesting this gut-cartilage axis plays a role in knee joint homeostasis4. In clinical populations, an analysis of the Rotterdam cohort revealed a significant positive relationship between joint pain and abundance of streptococcus species (spp.) in the gut microbiome, a finding that was replicated in the Lifelines-DEEP study6. This association was thought to be driven by local inflammation within the knee joint. However, because OA patients often have multiple co-morbidities7, there is a need to determine the disease-relevant gut microbial changes that are specifically involved in the pathogenesis of cartilage damage, while controlling for other factors.The gut microbiome has been implicated in the pathogenesis of both rheumatoid arthritis and osteoarthritis (OA)8. Therefore, interrogating relations between outcomes of interest in the absence of a host-response from adipose tissue provides a direct approach for investigating the role of gut microbiota in diseases processes. Using a mouse model of lipodystrophy (LD), we demonstrated that many factors previously associated with the obesity-induced OA  are indeed separable, as LD mice are protected from cartilage damage, and high fat feeding does not reverse the cartilage protection phenotype or worsen the metabolic dysregulation of LD mice fed a control diet9. Furthermore, cartilage protection in LD mice can be reversed by the implantation of a small transplant of adipose tissue. In this respect, comparing the gut microbiota across these conditions provides the opportunity to assess alterations in the gut microbiome while controlling for adipose tissue host-responses, obesogenic diet, and surgical onset of OA.Obesity is associated with several factors that potentially contribute to OA, such as gut microbiota dysbiosis, host responses from adipose tissue, circulating inflammatory mediators, and diet. However, the relative contributions of these factors in OA pathogenesis have been difficult to separate. The gut microbiota and its metabolites are key regulators of systemic inflammation from adipose tissue and are involved in the onset and entrenchment of metabolic disturbance9 using 16S rRNA sequencing of the fecal microbiota in an attempt to separate factors previously associated with OA and obesity  to evaluate relations between the gut microbiota and cartilage damage. We hypothesized that taxonomic changes in the gut microbiota are associated with OA severity when controlling for the effects of an obesogenic diet and of the presence of adipose tissue using LD mice. To add mechanistic insight to the evaluation of relations between the gut microbiota, the reintroduction of cartilage damage, and the absence of adipose tissue, we interrogated circulating and local levels of endotoxin, or lipopolysaccharide (LPS) in these animals10. This experimental design allows for the identification of gut microbiota changes that are associated with the reversal of cartilage protection and the onset of cartilage damage in this context.In the present study, we evaluated a subset of our previously reported cohortAll experimental procedures were carried out in accordance with the guidelines prescribed by the Washington University in St. Louis School of Medicine Department of Comparative Medicine, in compliance with the ARRIVE guidelines, and were approved by the Institutional Animal Care and Use Committee at Washington University in St. Louis School of Medicine.11. DTA/ + wild-type (WT) littermates were maintained as controls. Male and female mice were fed either a chow control diet  or high fat diet  from weaning. Further methodological details of this study can be found in the original paper detailing the cartilage phenotype of these mice9. Briefly, mice of different genotypes were co-housed to control for cage effects in gut microbiota composition. A subgroup of LD mice received either a mouse embryonic fibroblast transplant (MEF-R) between 3–5 weeks of age that forms an adipose-like tissue, or wildtype fat transplant containing visceral and subcutaneous fat (WF-R) between 6–8 weeks of age . At 16-weeks, mice underwent surgery for destabilization of the medial meniscus (DMM) on their left knee, and the right limb served as a non-surgical contralateral control9. Intact lower limbs, serum, synovial fluid and a single fecal pellet from the rectum were collected immediately after sacrifice at 28 weeks of age9. To evaluate cartilage damage severity, safranin-O/fast green stained sections were assessed using the Modified Mankin Score, which have been reported previously9. For the present study, serum and synovial fluid LPS concentration was measured using a Pierce LAL Chromogenic Endotoxin Quantitation Kit (Thermofisher Scientific).LD mice were created by crossing adiponectin-Cre mice with homozygous lox-stop-lox-ROSA-diphtheria toxin A (DTA) micewww.qiime2.org) and12 classified using BLASTn against the NCBI database. Species richness, or the total number of unique OTUs within each sample, and Shannon Diversity Index, a measure of microbiome richness and evenness, was calculated using standard methods13 in R version 1.2.1073 .Fecal samples were analyzed using 16S rRNA amplicon sequencing to evaluate the microbial ecology of each sample on the Illumina NovaSeq Platform . Further details can be found in Supplementary Methods. Operational taxonomic units (97% OTUs) were assigned using QIIME2  was utilized to determine significant differences in microbial community β-diversity. MaAslin2 was used to determine multivariable associations via generalized linear regression between taxonomic relative abundance and cartilage damage, operationalized by Modified Mankin Scores, by controlling for fixed effects of diet (chow or HFD), genotype (LD or WT), surgery and OA and random effect of individual samples14 with AST transformation of microbial abundance. LPS concentrations were analyzed by repeated measures ANOVA  and Tukey or Sidak post hoc testing with Graphpad Prism version 9 . We did not observe any significant differences in LPS levels, OA Scores, or microbiota Microbial taxonomic analysis was also performed using R version 4.0.2 and R studio version 1.3.1073 9.There was no difference in serum LPS levels among groups Fig. A. SynoviOTU richness was decreased in all groups compared to chow-fed WT animals Fig. C. SimilaANOSIM of the weighted UNIFRAC distance indicated significant differences between each treatment group with all other treatment groups (Supplementary Table paraprevotella spp. (Bacteroidetes phylum), Citrobacter spp. (Proteobacteria phylum), dorea spp. (Firmicutes phylum), Anaerovorax spp. (Firmicutes phylum), Blautia spp. (Firmicutes phylum), Mucispirillum (Deferribacteres phylum), and Lachnoclostridium spp. (Clostridium phylum). Significant positive associations were observed with Rikenella spp. (Bacteroidetes phylum) and Kingella spp (Proteobacteria phylum). A much larger array of significant associations was found between the other fixed effects, specifically by diet  in adipocytes and other host cells leading to systemic inflammation and metabolic disturbance, which may contribute to OA16. Furthermore, the gut microbiota may be influencing the joint more directly, or in the absence of a host responses through LPS or adipose tissue1. Within the joint, LPS is thought to prime pro-inflammatory macrophages through TLR-4 and is associated with activated synovial macrophages and knee OA severity10.Endotoxemia has been hypothesized as a key host-response linking gut microbiota changes and OA1. However, the sample size for WF-R SF was restricted due to the secondary nature of this analysis, and SF samples available from MEF-R were not available, so our interpretation of the role of local LPS is this disease process is inherently limited. Furthermore, in the present analysis, we did not measure earlier timepoints where LPS levels could have fluctuated and influenced the cartilage damage that we ultimately measured at 28-weeks. Our future work will detail time course changes in synovial fluid of all groups considered here.We did not observe differences in circulating levels of LPS between groups, suggesting that in this model system, systemic LPS is not associated with cartilage damage. However, we did observe local increases in LPS in the synovial fluid in HFD WT and partially in WF-R. Because WF-R LPS levels remained similar to that of chow-fed WT, chow-fed LD, HFD LD, and HFD-fed WT, and there were no increases in LPS in the DMM vs. non-surgical limbs, the relative contribution of SF LPS in the onset and progression of joint pathology is unclear. While it is possible that SF LPS plays an antagonistic role in joint damage we observed in HFD WT and the reversal of cartilage protection in WF-R, we would hypothesize that LPS levels would be increased in DMM limbs. More work is needed to make these determinations. Taxonomic changes we measured in this model may be more directly linked to cartilage damage, and perhaps not reliant on a host response (i.e. from adipose tissue). This also may provide partial support of the hypothesis that the gut bacteria-cartilage axis can be direct and not necessarily dependent on LPS signaling or host-responses in adiposeAnaerovorax spp. are typically depleted in obese individuals, which is consistent with the significant negative relationship found here with OA when controlling for diet17. While the association between Rikenella spp. and cartilage damage is a novel association, Kingella spp. are common causes of septic arthritis and osteomyelitis in children18. Rikenella spp. and Kingella spp. are gram-negative species, so if they are indeed migrating to the joint through the circulation, could be contributing LPS within the joint directly10. Blautia spp. and Dorea spp. are known to produce butyrate, an anti-inflammatory metabolite that has been shown to modulate inflammation in chondrocytes through G Protein-coupled Receptor 43 (GPR43)19. While Streptococcusspp. were not found to be independently associated with OA when controlling for other factors as has been observed in other studies, we did observe a strong positive correlation with high fat diet5. It is possible that Streptococcus spp. may elicit a host response through adipose or other tissues that is not observed in the fat-free lipodystrophic mice, but future work is necessary to directly test this speculation.Several of the novel associations noted here are concordant with previous reports in the literature. 20, supporting the notion that modification of the gut microbiome could provide an attractive, minimally-invasive therapeutic target for cartilage damage22. For example, germ free mice demonstrate less bone loss in response to non-invasive load-based injury compared to conventional controls23. When germ free mice are challenged with DMM, the same injury used in the present study, they demonstrate significant reductions in osteophyte size and improvements in cartilage damage compared to conventional control mice, illustrating a role for the gut microbiota in promoting OA24. Furthermore, antibiotic treatment prior to injury has been shown to mitigate OA caused by a loading-induced injury25, as well as in toll-like receptor 5 knockout mice that are protected from metabolic syndrome26. Antibiotic-induced dysbiosis from treatment with ampicillin and neomycin reduced serum levels of LPS and improved OA outcomes after injury27, further illustrating that ablation of certain microbes could be a viable preventative treatment strategy for cartilage damage. Associations between the gut microbiome, muscle quality28, and bone strength29, contributors to OA pathogenesis30, have also been identified, suggesting that modulating the microbiome could have broad protective applications on the joint organ system. The present study contributes new knowledge to the identification of potential targets within gut microbiota for modification. It is possible that prebiotic fiber, probiotic supplementation, or repletion and/or selective depletion of the microbial taxa reported here could override cartilage protection LD mice, and future studies aim to determine cause-effect relationships in this context32.Mitigating alterations in the composition of the gut microbiota using prebiotic fiber and exercise have been demonstrated to be protective from OAThis study has several potential limitations. By design, this study is a cross-sectional secondary analysis, and as such, we cannot deduce specific taxonomic changes involved throughout the progression of cartilage damage. Our synovial fluid analysis is limited in sample number and lacks the MEF-R group, so we cannot completely evaluate the potential increase in LPS and its role in the reversal of cartilage damage in fat rescued mice. Future efforts will chronicle time-course changes in fecal microbiota, synovial fluid, and serum after injury in LD, WT, and fat-rescued mice to understand taxonomic and endotoxin changes as they may occur with worsening joint damage. Furthermore, the LD mouse model is a well-controlled model system lacking adipose tissue, but as such, the insights identified here are context-dependent and need to be validated in other preclinical model systems. Finally, our use of 16s sequencing limits the interpretation of the present dataset to taxonomic profiling. Future work will employ shotgun metagenomic sequencing to interrogate metabolic function profiling.Rikenella spp. (Bacteroidetes phylum) and Kingella spp (Proteobacteria phylum). The significant negative associations include: Paraprevotella spp. (Bacteroidetes phylum), Citrobacter spp. (Proteobacteria phylum), Dorea spp. (Firmicutes phylum), Anaerovorax spp. (Firmicutes phylum), Blautia spp. (Firmicutes phylum), Mucispirillum (Deferribacteres phylum), and Lachnoclostridium spp. (Clostridium phylum). This analysis identified targets that can be precisely modulated in future studies, colonizing germ free mice with specific microbial consortia, and/or selective addition or depletion of taxa within a community to understand the causal role between taxonomic alterations in the gut microbiome and cartilage health.In conclusion, this novel murine model of lipodystrophy can be used to separate the role of diet, adipose tissue host-response, and systemic inflammation, allowing analysis of links between gut microbiota and cartilage damage. We identified 9 novel associations between gut microbiota species and Modified Mankin Scores when controlling for inflammation and diet. The significant positive associations include: Supplementary Information.


Introduction Brachial plexus lipomas are extremely rare benign tumors that may cause slow progression of neurological deficits leading to thoracic outlet syndrome. Up to now, surgery remains challenging. The aim of this study is to present our surgical treatment regime and long-term neurological outcome in three cases of giant brachial plexus lipomas and to show results of systematic review.Patients and Methods Retrospective analysis of our database “peripheral nerve lesion” to identify patients suffering from brachial plexus lipomas between January 1, 2012, and December 31, 2019. Systematic review was performed for literature published until March 31, 2020, analyzing PubMed, Google Scholar, Scopus, and the Cochrane Collaboration Library independently by two authors.Results Over the past years, three patients suffering from giant brachial plexus lipomas attended to our neurosurgical department. All patients underwent preoperative magnetic resonance imaging (MRI), ultrasound examinations, and electrophysiological testing. Tumors were removed microsurgically via anterior/posterior, supraclavicular/infraclavicular, and combined approaches. The patients were accessed postoperatively by MRI and clinical follow-up. Systematic review of the literature revealed 22 cases, which were analyzed in regard to demographics, surgical treatment, and neurological outcome.Conclusion Brachial plexus lipomas are an extremely rare cause for brachial plexus compression. Total microsurgical removal with intraoperative electrophysiological monitoring is the treatment of choice with excellent long-term MRI and clinical outcome. Brachial plexus tumors are relatively rare, and treatment is often challenging due to the complex anatomical location with adjacent nervous and vascular structures. In the majority of the cases, lipomas are benign asymptomatic soft tissue tumors showing only slow progression. Nevertheless, in very rare cases, deep-seated lipomas can grow to gigantic size measuring more than 10 cm of lengthThis report presents our microsurgical treatment regime of brachial plexus lipomas with intraoperative electrophysiological monitoring and ultrasound assistance. Furthermore, systematic review was performed.Retrospective analysis of our database “peripheral nerve lesion” revealed three cases of large brachial plexus lipomas all causing neurogenic TOS (nTOS). We analyzed medical records and preoperative/postoperative magnetic resonance imaging (MRI) and performed follow-up examinations to evaluate the long-term outcome.The study was approved by the Institutional Review Board .Systematic review was performed for literature published until March 31, 2020, analyzing PubMed, Google Scholar, Scopus, and the Cochrane Collaboration Library independently by two authors. Search keywords comprised “lipoma,” “brachial plexus, brachial plexus neuropathies or brachial plexus neuropathy.” Inclusion criteria were articles published in English presenting the clinical course, peripheral nerve location, and the treatment regime.Furthermore, the reference lists of included articles were reviewed to identify and include additional eligible articles. Those studies were meticulously cross-referenced to ensure that patients were not included in multiple articles . The syA 62-year-old male patient presented with a pectoral subcutaneous mass lesion. The tumor showed progressive and visible growth over a period of more than 7 years, finally causing atrophy and a severe paresis of the triceps brachii muscle, the biceps brachii muscle, the deltoid muscle, and the supraspinatus muscle. Electrophysiological testing revealed an impairment of the left brachial plexus and MRI showed a large lipomatous tumor extending from the area of the triceps brachii muscle toward the supraspinatus muscle, the clavicle, and the brachial plexus on the left side (8 × 7 × 6 cm). Total piecemeal tumor resection via a two-portal supraclavicular and infraclavicular approach and neurolysis finally led to a slight improvement of the paresis on the follow-up examination 12 months after surgery .A 61-year-old male patient presented with a 10-year history of right-sided TOS causing hypoesthesia of digitus IV and V, a severe paresis of the triceps brachii muscle, the biceps brachii muscle, and the dorsal and palmar interossei muscles. Ultrasound and MRI of the neck depicted a large lipomatous mass (10 × 7 × 5 cm) compressing the right brachial plexus. Electrophysiological testing showed impairment of the axillary nerve. Total tumor resection via a dorsal suprascapular, infraclavicular approach and neurolysis finally led to improvement of the hypoesthesia 4 weeks after surgery. The severe paresis did not improve until the first neurological follow-up 15 months after surgery .A 56-year-old female patient presented with a progressive swelling of the left neck and shoulder over a period of around 2 years. Preoperative MRI revealed a left-sided brachial plexus lipoma with contact to the brachial plexus. Total tumor resection finally led to long-term relief of the symptoms. Follow-up examination 9 months after surgery revealed no neurological deficit.The patient was positioned in supine position and ultrasonography showed the supra- and infraclavicular lipomas under the pectoral muscle. Skin incision along the pectoral muscle to the axilla was followed by subcutaneous preparation toward the fascia of the muscle. Further preparation in the infraclavicular fossa visualized the lipoma beneath the clavicula. The capsule of the lipoma was then mobilized microsurgically. Further preparation was achieved via a second skin incision above the clavicula. Bipolar electrical stimulation  was performed at the medial parts of the lipoma to test each nerve structure proximally and distally with voltages from 2 to 5 mA showing a vivid motor response. Mobilization of the tumor capsule was followed by piecemeal removal of the lipoma toward the brachial plexus using the Sonoca 300  after nerve stimulation. A hemostasis procedure and subsequent removal of the lesion were followed, using bipolar coagulation. At the end, total tumor resection was achieved, and electrophysiological nerve stimulation showed that the displaced brachial plexus was not harmed .Database search revealed 450 articles. Total 382 articles were excluded after analyzing the title and the abstract. The full text of the remaining 68 articles was reviewed and 58 articles were excluded afterward. In total, 10 articles were included,Three variants  of TOS have been described depending on the underlying pathophysiology.An extremely rare cause for nTOS is large lipoma adjacent to the brachial plexus, usually presenting as a subcutaneous mass and upper limb or shoulder pain. Lipomas are benign, slow-growing soft tissue tumors and the most common mesenchymal tumor with an estimated incidence of 10%.They can arise adipose tissue in any part of the body. Lipomas are commonly located in the back, thigh, forearm, chest, and the posterior neckLipomas should be differentiated from other brachial plexus tumors. Most common benign brachial plexus tumors are schwannomas and neurofibromas.Nonoperative and surgical approaches have been described as therapy of TOS.Lipomas adjacent to major nerves and vessels should be removed by microsurgical excision with electrophysiological neuromonitoring to prevent neurological deficits. Blind aspiration may injure these structures, resulting in a major bleeding or neuromuscular dysfunction with paralysis.Although advances in surgical strategies including peripheral nerve microsurgery have improved the outcome, there is still a risk of causing intraoperative damage. Preserving the neurological function while trying to achieve gross total resection should be the principle treatment goal. Therefore, intraoperative neurophysiological monitoring combined with intraoperative high-resolution ultrasound represents an effective technique for identifying and monitoring functional integrity of both the spinal cord and the nerve roots in real time. The benefit of intraoperative neurophysiological monitoring for preserving neuronal structures and achieving an optimal postoperative functional outcome was proven in several studies.In our opinion, microsurgical resection with intraoperative electrophysiological monitoring should be performed to remove the tumor in total and to avoid nerve injuries.Brachial plexus lipomas are an extremely rare cause for TOS and are typically diagnosed several months delay after the initial symptoms. Microsurgical removal under permanent intraoperative electrophysiological monitoring should be the treatment of choice as it is safe and has a favorable outcome.


Internal ribosomal entry sites (IRESs) are RNA secondary structures that mediate translation independent from the m7G RNA cap. The dicistronic luciferase assay is the most frequently used method to measure IRES-mediated translation. While this assay is quantitative, it requires numerous controls and can be time-consuming. Circular RNAs generated by splinted ligation have been shown to also accurately report on IRES-mediated translation, however suffer from low yield and other challenges. More recently, cellular sequences were shown to facilitate RNA circle formation through backsplicing. Here, we used a previously published backsplicing circular RNA split GFP reporter to create a highly sensitive and quantitative split nanoluciferase (NanoLuc) reporter. We show that NanoLuc expression requires backsplicing and correct orientation of a bona fide IRES. In response to cell stress, IRES-directed NanoLuc expression remained stable or increased while a capped control reporter decreased in translation. In addition, we detected NanoLuc expression from putative cellular IRESs and the Zika virus 5′ untranslated region that is proposed to harbor IRES function. These data together show that our IRES reporter construct can be used to verify, identify and quantify the ability of sequences to mediate IRES-translation within a circular RNA. All eukaryotic mRNAs are co-transcriptionally capped with a m7G cap . This caViruses employ specific strategies to take over the translation machinery in cells. Poliovirus and other viruses from the Picornaviridae family express viral proteases that cleave translation initiation factors eIF4G and polyA-binding protein (PABP), which prevents the translation of the majority of mRNAs ,12,13. HBecause cellular mRNAs are always capped, the question of whether cellular mRNAs can have an IRES was heavily debated . SpecifiRenilla and firefly luciferases, in a sequential orientation with an IRES or a putative IRES in between the ORFs. Following transcription of this plasmid, the Renilla luciferase in the first cistron is translated via the canonical cap-dependent translation initiation pathway. In contrast, the firefly luciferase ORF is translated via an IRES. Although this assay is straightforward, great care must be taken to rule out false-positive results due to the presence of cryptic promoters, cryptic splice sites, and by ribosome readthrough [To determine if an RNA contains an IRES, a dicistronic luciferase assay is typically employed . Such pldthrough . CrypticRNA circularization has been proposed and shown to be an alternate more accurate approach to test for IRES activity . CirculaIt was shown that the full-length intronic repeats of the ZKSCAN1 gene can mediate RNA circularization ,28. TheyA backsplicing reporter plasmid pcDNA3.1(+) ZKSCAN1 MCS-WT Split GFP  was purcFor the EMCV reverse IRES control and the Myc, DAP5 and c-Jun sequences, sequences were PCR-amplified using Q5 DNA polymerase  using DNA ligase buffer (NEB), annealed and inserted into the ZKSCAN split NanoLuc plasmid.The N- and C-terminal halves were individually amplified from the ZKSCAN plasmid and inserted into the HindIII and BamHI restriction sites of pcDNA3.Ligations were performed using T4 DNA Ligase (NEB) following the manufacturer’s instructions.For the donor and the acceptor splice site mutants, PCR and KLD treatment were performed, as described in the Q5 mutagenesis kit (NEB).For insertion of the ZIKV UTRs, two gene blocks containing the ZIKV 5′-3′ UTRs and 3′-5′ UTRs were purchased from GENEWIZ/Azenta Life Sciences. These sequences and the single 5′ UTR and the 3′ UTR were PCR amplified and ligated into the SbfI and EcoRV sites of the ZKSCAN split NanoLuc plasmid.To generate the dual-luciferase construct, the firefly luciferase was amplified from pGL4.13 plasmid and inserted into the NheI and BamHI sites of the linker and the c-Myc IRES-containing ZKSCAN NanoLuc plasmids.All constructs were transformed into DH5α cells and grown overnight on LB-Carbenicillin plates. Following miniprep plasmid isolation, the correct construct was confirmed by Sanger sequencing.v/v) FBS , 2 mM L-glutamine .Adherent HeLa S3 cells were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% , following the manufacturer’s protocol. Cells were grown for 24 h, then lysed in passive lysis buffer . Firefly luciferase and NanoLuc activities were measured in a Glomax instrument using the Dual Nano-Glo kit (Promega).IRESs are well-known to mediate translation through a cap-independent mechanism and the dicistronic luciferase reporter system is the gold standard to measure IRES-mediated translation. However, the dual-luciferase system can yield false-positive results and hence requires several control experiments to deduce the correct data. To overcome such issues, we measured translation from a circular RNA generated from a new backsplicing reporter construct as an alternative approach. While generating circular RNAs by splinted ligation suffers from low yield, recent research has shown that cells can synthesize RNA circles by a backsplicing event that is facilitated by complementary sequences upstream and downstream of the RNA sequence of interest included in the circle. In this study, we used a previously published plasmid (pcDNA3.1(+) ZKSCAN1 MCS-WT Split GFP) to develop a quantitative circular RNA reporter . InsteadFor the split NanoLuc reporter, we sought to split the NanoLuc ORF in a position where both NanoLuc fragments, when co-expressed, would not be able to yield active NanoLuc protein. Zhao et al. showed that a NanoLuc protein split between amino acid positions 117 and 118 is unable to complement and yield active NanoLuc . Hence, Following co-transfection of the ZKSCAN EMCV split NanoLuc and the pGL4.13 plasmid encoding a firefly luciferase protein, both NanoLuc and firefly luciferase activities were measured and the NanoLuc over firefly ratio was plotted B. To shoThe EMCV IRES is a strong viral IRES that is frequently used as a positive control for IRES-mediated translation. Since many positive-sense viral RNA genomes do not use the typical m7G cap, and these viruses often express proteins that disrupt cap-dependent translation by cleaving translation initiation factors, functional IRES sequences were easily proven. In contrast, cellular mRNAs are always capped during synthesis, which has hampered research into their ability to mediate cap-independent translation. To test various cellular IRESs’ ability to mediate translation from a circular RNA, we PCR-amplified and ligated the putative cellular IRES sequences from c-Myc, DAP5 and c-Jun into the split NanoLuc reporter A. IRES aOne of the best-studied viral IRESs is the IRES from hepatitis C virus, which is a member of the Flaviviridae family. Although members of the genus flavivirus have been shown to use an RNA cap structure, there is evidence from the Dengue virus that these viruses may also use a cap-independent pathway for translation initiation . More reTo eliminate the need for co-transfection of the firefly luciferase report and improve ease of use, we next generated a reporter that encoded both firefly and NanoLuc proteins on the same mRNA A. We PCRIn this study, we replaced the split GFP within the pcDNA3.1(+) ZKSCAN1 MCS-WT Split GFP plasmid with a split NanoLuc gene to create a quantitative and sensitive reporter for circular RNA translation. We showed that an IRES and backsplicing are required to produce NanoLuc. We further tested the translation of putative IRES sequences from cellular sequences and ZIKV. Lastly, we inserted a firefly luciferase ORF upstream of the split nanoluciferase gene. This dual-luciferase construct no longer requires co-transfection of two plasmids, as cap-dependent and cap-independent reads can be measured from the same plasmid.To overcome limitations of the traditional dicistronic luciferase assay, circular RNAs have been proposed to accurately measure IRES-mediated translation  with celWe further showed that, upon insertion of putative cellular IRES, NanoLuc activity could be measured. The c-Myc mRNA is known to be translated cap-independently, and extensive testing had been performed ,35,43. SOne feature of viral and cellular IRESs is that their translation continues during induction of the intergenic stress response and phosphorylation of eIF2α . SimilarWe additionally used the split NanoLuc reporter to test another putative IRES, the Zika virus IRES. It had been reported that the Dengue virus could be translated cap-independently, although it was initially proposed that this mechanism was not IRES-mediated . SeveralFor our initial co-transfection experiments, we employed two constructs, each with a different promoter  to avoid promoter competition. However, we did notice variability in the data, likely due to unavoidable transfection efficiency. We generated and tested the transfection of a dual-luciferase version of the reporter to reduce variability. In this construct, the expression of NanoLuc and firefly luciferase is mutually exclusive with the linear transcript responsible for firefly luciferase expression, while the NanoLuc is only expressed upon backsplicing. Interestingly, the insertion of the firefly luciferase upstream reduced NanoLuc production in the plasmid containing the c-myc IRES, suggesting that insertion of an upstream sequence could influence the backsplicing rate and hence NanoLuc levels. Although our construct is the first utilizing luciferase enzymes, a similar construct encoding a Ruby Red fluorescent protein to measure cap-dependent translation and the split GFP to measure circular RNA translation has been used to perform a screen for putative IRESs . From 55Circular RNAs have advantages over the dicistronic luciferase assay to measure IRES-mediated translation. Here, we adopted a previously published ZKSCAN split GFP to create a split NanoLuc construct. This split NanoLuc construct is another tool to help investigate circular RNA IRESs further and measure and compare their ability to mediate translation using a sensitive luminescence assay.