To date, urethral stricture disease in men, though relatively common, represents an often poorly managed condition. Therefore, this article is dedicated to encompassing the currently existing data upon anatomy, etiology, symptoms, diagnosis, and treatment of the disease, based on more than 40 years of experience at a tertiary referral center and a PubMed literature review enclosing publications until September 2018. Urethral stricture disease can develop throughout the entire length of the male urethra and can be caused by a large variety of etiologies. Also, urethral strictures give rise to a wide range of symptoms and warrant a specific diagnostic work-up before proceeding to any treatment modality.th century, urologists have attempted to find solutions to treat both simple and complex urethral strictures and, over the last few decades, research has mainly been focused on refining the existing procedures to mitigate the negative postoperative consequences. However, despite the substantial scientific progress on this subject, a numerous amount of studies has revealed the insufficient knowledge about urethral stricture surgery among urologists and has shown that patients with urethral strictures are generally offered an inadequate treatment option . In. In63]. (2) Free Graft Urethroplasty. From the moment a stricture is no longer an indication for end-to-end urethroplasty, a substitution urethroplasty is unavoidable [voidable . Herein,voidable . Variousvoidable . Furthervoidable . Most liPreputial Grafts versus Buccal Mucosa Grafts. Nowadays, the trend is to use buccal mucosa grafts over preputial grafts  Pedicled Flap Urethroplasty. Pedicled flaps carry their own vascular supply in the pedicle and therefore can survive independent of the surrounding tissues. The preputium  Multistage Urethroplasty. Given the outstanding success rates of one-stage urethroplasty, the indications for multistage procedures have diminished remarkably. Nowadays, staged interventions are mostly reserved for redo cases in which there's a complete lack of healthy tissue and only very precarious vascularization.Almost every multistage urethroplasty technique is derived from the original Johanson technique . The genAfter Johanson, several surgeons—Turner-Warwick , Gil-Ver(5) Mesh Graft Urethroplasty or Multistage Oral Mucosa Graft Urethroplasty. Mesh graft urethroplasty and multistage oral mucosa graft urethroplasty both represent a variation of multistage urethroplasty in which the disadvantages of tubularizing hairy skin segments  are avoided. With the mesh graft urethroplasty, a split thickness skin graft is harvested using a dermatome and meshed, and, with the multistage oral mucosa graft urethroplasty, a piece of oral mucosa is harvested and prepared as described above. The mesh graft or oral mucosa graft is then sutured in between the created urethral edges (after opening of the urethra) and the borders of the skin and, subsequently, during the second stage, there is no need to tubularize hair bearing skin, but only hairless graft material. These techniques are mostly reserved for complex reconstructions and mesh grafts are particularly interesting in cases with restricted graft/flap options [ options , 93.(6) Definitive Perineostomy. Nowadays, multistage urethroplasty is almost exclusively preferred in rather complex cases and in patients that have had numerous urethral interventions already. Often, these patients are perfectly happy to void without any problems after the first stage of an intended multistage procedure and wish to retain the created perineostomy without a second-stage procedure. This definitive perineal urethrostomy represents a well-accepted situation for many patients, especially multioperated and older ones.For the creation of a definitive perineostomy, the Johanson and Blandy techniques are most frequently used and entail similar success rates . With th(7) Tissue-Engineering in Urethroplasty. Urethral reconstruction using matrix-bred tissue out of the patient's own urothelium or oral mucosa has recently been introduced as an alternative approach which mainly addresses the limitations inherent to the classic substitution urethroplasty. So far, tissue engineering with urothelium has been described in laboratory studies [2 oral mucosa biopsy was harvested and sent to the laboratory for tissue engineering. After 3 weeks, the manufactured piece of tissue was sent back to the hospital and administered during urethroplasty . studies , 97 and, studies . In theiToday, the largest limit of tissue engineering lies within its cost, but, definitely, this technique involves several advantages, especially when the classic substitution materials become scarce or even totally absent. Furthermore, it reduces the amount of donor tissue that is required for reconstruction and could therefore potentially reduce the side-effects of the graft site that are seen after a classic free graft urethroplasty . Nonethe(8) Combination of Techniques. In case of multiple urethral strictures or very long, often complicated urethral strictures, a combination of the aforementioned techniques might be necessary to offer the patient a one-stage solution [solution , 100. InFor a primary urethroplasty, the surgeon should always opt for the simplest and most straightforward technique that yields the highest success rates. Ideally, the chosen technique also represents the treatment option that least compromises the therapeutic armamentarium that might be needed in the future as there is always a risk for urethroplasty failure requiring one or more salvage treatments .For isolated, short (≤3.0 cm), bulbar urethral strictures, the authors follow the end-to-end urethroplasty recommendation of the ICUD, although a lot of controversy exists as mentioned above , 49, 50.The more redo procedures a patient has undergone, the harder it gets to choose between surgical approaches, as the options become sparser and gradually less favorable. In these cases, it is hard to stipulate general rules as these treatment decisions are very case-specific and need to be well deliberated. In long, complex and multioperated urethral strictures, a preputial or scrotal skin flap could remain a viable option, at least if this tissue is still available. An alternative is to perform a multistage urethroplasty or to construct a definitive perineostomy, which is often well-tolerated by patients with an elaborate history of urethral stricture treatments.It should be underlined that this decision-making process may not be universal and that there is a lot of controversy about treatment algorithms for urethral reconstruction, especially in the bulbar urethra , 49, 50.(1) Posterior Urethral Strictures. These strictures are characterized by the following:An anatomical proximity to the urethral sphincter and possible extension deeper than the urogenital diaphragmA distraction defect between the prostatic apex and the distal urethral portionConcomitant hematoma formation, infection, and/or previously failed urethral realignment procedures (endoscopic or open) which might lead to extensive stricture formation in the entire zone between both disrupted urethral ends, which makes it hard to recognize the local anatomy. Furthermore, in strictures related to a pelvic fracture, the displacement of bony fragments might hamper the exposure, which further complicates the surgical procedureIn traumatic strictures, the possibility of coexisting damage to the penis, scrotum, and/or perineum, which may heavily compromise the available surgical options to restore the urethral continuity The standard approach after trauma related urethral injuries involves the immediate placement of a suprapubic catheter  followed(2) Meatal Strictures. Meatal strictures may be located only at the meatus urethrae but can also expand in the navicular fossa or the entire transglandular segment. In some cases, the meatal stricture is part of an entire diseased anterior urethra.A meatal stricture is often considered as a minor and rather benign condition, although, in every case, the clinician has to consider the possibility of underlying lichen sclerosus. Furthermore, obtaining a perfectly functional and esthetic result is harder than it may seem. Herein, the severe deviation of the urinary stream represents the most bothersome complication as it will obligate the patient to void in a sitting position.The true extent of a meatal stricture is often difficult to estimate preoperatively. In pronounced strictures, a RUG will usually be impossible to perform and, during a VCUG, the entire proximal urethral segment will dilate but will only poorly reveal the true length of the stricture. In fact, the most reliable length measurement takes place in the operating theatre. Hence, the surgeon starting the meatoplasty should always master a variety of techniques as every patient will need an individualized surgical approach.Several techniques have been described to reconstruct the urethral meatus . Many ofThe use of genital skin must be avoided in all patients with a lichen sclerosus related stricture as it can lead to a failed procedure ; in thes(3) Lichen Sclerosus Related Strictures. Lichen sclerosus is a chronic, inflammatory skin condition with a specific predilection for the genital region. Furthermore, this disease may importantly affect the penile as well as the bulbar urethra [ urethra , 9. ThesAs mentioned above, it is generally accepted that lichen sclerosus related strictures are not to be treated with skin as substitution material, but with oral grafts . NonetheBased on good dermatological results after high-dosed, locally applied corticosteroids, its value has also been evaluated for intra-urethral administration and has shown satisfying results in small patient series , 107. It(4) Urethral Strictures after Local Prostate Cancer Treatment. All local treatment options for prostate cancer  or cryotherapy) may contribute to subsequent stricture formation, located between the bladder neck and bulbar urethra [ urethra . These s urethra –112. The urethra . Substit urethra . HoweverIt must be acknowledged that, overall, these patients are hard to treat and that urethroplasty in these cases holds an important risk for failure. Furthermore, the presence of a stricture may have concealed a problem of underlying incontinence, which may suddenly appear after a successful procedure in which the urethral patency has been restored. Hence, all patients should be thoroughly informed preoperatively and well counseled about the potential consequences of treating their stricture.In a literature review incorporating 36 studies with a total of 2323 patients, persisting de novo erectile dysfunction has been described in 1% of the patients after urethroplasty . This nuWith the classic transecting end-to-end urethroplasty, the erectile tissue is directly damaged during surgery and thus one could expect that erectile function importantly decreases after this procedure. This assumption has been illustrated by a study of Ekerhult et al., although they could only withhold a 5% incidence rate of de novo erectile dysfunction after end-to-end procedures . Herein,Ejaculatory function is often better after urethroplasty than before, provided the use of a technique in which the continuity of the bulbospongiosus muscle is actively restored during the multilayered closure of the perineum , 120.After urethral reconstruction, it is important to provide urinary derivation as urine extravasation at the recently operated region might lead to important complications, such as abscess formation and phlegmon. When a free graft has been used, the authors routinely leave a 20 Fr transurethral catheter in place to avoid prolapse of the graft into the urethral lumen and to allow close contact between the graft and its vascular bed. In other cases, urinary derivation is assured through a 16 Fr transurethral catheter or a suprapubic tube. Nonetheless, it should be underlined that catheter use after urethral reconstruction is extremely variable between different urethroplasty centers, without clear data if one regimen is truly better than the other.Most patients are discharged from the hospital on the second or third postoperative day with the indwelling catheter in place. At that moment, instructions for wound care are provided, which are specifically important in patients with a perineal wound. These wounds need to be kept dry and clean. Therefore, the use of a hair dryer (3 to 4 times a day) and repeated disinfection is advised, a method that was copied from wound care principles after episiotomy in females.Routine perpetuation of the antibiotic treatment regimen must be limited to those patients in which a preoperative urinary tract infection has been established. In these cases, appropriate antibiotics (according to the antibiogram) are continued for a maximum of 5 days since any longer use will only contribute to the problem of resistant microorganisms. There is no clear evidence to support this advice in urethroplasty, although the authors base this recommendation upon the general rules and principles of antibiotic therapy.After 7 to 14 days, the first postoperative visit is scheduled and involves the execution of a VCUG, after filling the bladder with contrast medium through the indwelling catheter. Some authors argue the value of a routine postoperative urethrography as only few patients will show contrast extravasation requiring a catheter replacement , 122. HoTo date, the ideal follow-up of patients after urethroplasty remains poorly defined. At Ghent University Hospital, patients are scheduled to revisit after 3 months, after 12 months, and annually thereafter. During these visits, anamnesis, physical examination, uroflowmetry, and Urethral Stricture Surgery Patient Reported Outcome Measures (USS-PROM) questionnaires are routinely administered , 126. AdTo date, there is no clear consensus about standard administration of urethrography and/or urethroscopy during follow-up. There is, however, a remarkable trend to use patient-reported outcome measures (PROMs) after urethral reconstruction. In 2011, Jackson et al. created and validated the USS-PROM, a questionnaire specifically made for patients after urethroplasty . Later, The optimal follow-up schedule will need further elucidation in the future. Presumably, this will not be a story of “one-size-fits-all” as urethral stricture disease entails a very heterogeneous patient cohort which certainly demands patient-adapted follow-up strategies.Male urethral stricture disease embodies a very heterogeneous condition in which thorough knowledge about anatomy, etiology, symptoms, diagnosis, and treatment aspects is crucial in optimizing care of these patients. Future prospective research will be warranted to gain further evidence and to refine the current practice of managing male urethral stricture disease.


In this study, a soft structure with its stiffness tunable by an external field is proposed. The proposed soft beam structure consists of a skin structure with channels filled with a magnetorheological fluid (MRF). Two specimens of the soft structure are fabricated by three-dimensional printing and fused deposition modeling. In the fabrication, a nozzle is used to obtain channels in the skin of the thermoplastic polyurethane, while another nozzle is used to fill MRF in the channels. The specimens are tested by using a universal tensile machine to evaluate the relationships between the load and deflection under two different conditions, without and with permanent magnets. It is empirically shown that the stiffness of the proposed soft structure can be altered by activating the magnetic field. The development of flexible or soft structures based on smart materials has attracted considerable interest owing to their effectiveness for vibration control through the tunable viscoelastic properties by external stimuli such as magnetic fields. Electrorheological fluids (ERFs), magnetorheological fluids (MRFs), and magnetorheological elastomers (MREs) are some of the smart materials which are frequently used for the vibration control of flexible structures. The ERF is a special suspension consisting of fine non-conducting particles 5–50 μm) and a carrier liquid which is electrically insulated. The apparent viscosity of the ERF can be adaptively tuned by applying an electric field; its tuning capability is reversible. The response to a viscosity change is very fast 3–5 ms). Therefore, ERFs are frequently used as core components of sandwich structures  to achieve excellent controllability of modal characteristics by applying different electric fields to the fluid domain. Therefore, an effective vibration control is provided by switching the natural frequency based on the excitation frequency to avoid a resonance behavior ,2,3,4. S–5 ms. Th–50 μm anMost studies on soft or flexible structures have focused on vibration control or/and vibration suppression by using the inherent field-dependent characteristics of smart ERFs, MRFs, and MREs. On the contrary, no extensive studies have been carried out on soft or flexible structures for stiffness tuning or control. Wagner and Bauer  have sumIn this study, a flexible soft structure, whose stiffness can be tuned, is proposed, and its potential is validated by a test-bed experiment. The proposed soft structure is fabricated by using an MRF embedded in channels in thermoplastic polyurethane (TPU). Beam types of specimens with 12 channels and three channels, respectively, are fabricated by three-dimensional (3D) printing for both channel patterning and MRF injection with two nozzles. In the fabrication, a filament wire is used to provide sealing to avoid MRF leakage. A tensile test is then carried out to evaluate the relationship between the applied load and corresponding deflection. In addition, the stress–strain curve is measured without and under a magnetic field to evaluate the tuning range of the Young’s modulus of the proposed soft structure. The presented preliminary results demonstrate the stiffness tunability of the proposed soft structure, which can be further analyzed for various applications including the discrete-channel-control-based bio-haptic patch. Two types of specimen are fabricated, and their configurations and geometric dimensions are shown in As the first step, the outside wall is stacked by the polymer, followed by the filling of MRF. In this process, the deflection of the upper side of the wall is prevented by adjusting both the surface tension and movement speed of the nozzle. In addition, the sealing issue (to avoid MRF leakage) is overcome by using the thermal fusion method, in which the stacking sequence is completed with the adhesive on the TPU. The total volume to be filled in the 12 channels is calculated, and then the same amount of MRF is charged to avoid the cavity problem. Prior to the test, different approaches of magnetic field generation are considered, such as the use of a circuit core and use of a permanent magnet (PM). The former method is expected to be more effective to achieve a wider stiffness tuning range, but its practical realization is complex. Thus, a PM is used in this study to generate a magnetic field around the MRF domain. In this study, a new flexible beam structure with a tunable stiffness was fabricated by 3D printing. Its field-dependent stiffness was experimentally validated. To tune the stiffness, the MRF was embedded in the 12 channels (specimen 1) and three channels (specimen 2) in the skin of the TPU. The stiffness of specimen 1 could be increased by approximately 22.9% and 13.9% by applying a magnetic field to the MRF domain by using PMs attached on the top and bottom for 1 mm and 2 mm strokes, respectively. By changing the position of PMs, the stiffness increment can be altered. Therefore, the optimal locations of PMs need to be determined to maximize the stiffness increment. In the test of specimen 1, the stress–strain results showed that the stress of 110.74 kPa without the magnetic field was increased to 135.82 kPa by PMs attached at both sides of the specimen. On the other hand, in specimen 2, the average stiffness increment ratios from the origin to the strokes of 1 and 2 mm were identified as approximately 39.78% and 23.10% upon the application of the magnetic field, respectively. It is also noted that specimen 2 exhibits considerably a higher increment of stiffness than specimen 1.These preliminary results presented in this work are self-explanatory in justifying the proof-of-concept of the stiffness change of the proposed soft structure by applying the magnetic field. Further studies are required to develop advanced multifunctional soft structures, based on the use of thinner skins (1–2 mm) with diverse shapes of channels, to provide local and global stiffness tuning and to investigate the haptic function by activating each channel. This study can guide the development of practical products such as stretchable bio-patches which can stimulate specific abnormal nerves and soft robots with wide flexibility ranges.


Nonenzymatic nucleic acid amplification techniques  have shown promising potential for amplified detection of biomarkers. e.g. the hybridization chain reaction, HCR) have shown promising potential for amplified detection of biomarkers. However, the traditional HCR occurs through random diffusion of DNA hairpins, making the kinetics and efficiency quite low. By assembling DNA hairpins at the vertexes of tetrahedral DNA nanostructures (TDNs), the reaction kinetics of the HCR is greatly accelerated due to the synergetic contributions of multiple reaction orientations, increased collision probability and enhanced local concentrations. The proposed quadrivalent TDN (qTDN)-mediated hyperbranched HCR has a ∼70-fold faster reaction rate than the traditional HCR. The approximately 76% fluorescence resonance energy transfer (FRET) efficiency obtained is the highest in the reported DNA-based FRET sensing systems as far as we know. Moreover, qTDNs modified by hairpins can easily load drugs, freely traverse plasma membranes and be rapidly cross-linked via the target-triggered HCR in live cells. The reduced freedom of movement as a result of the large crosslinked structure might constrain the hyperbranched HCR in a confined environment, thus making it a promising candidate for in situ imaging and photodynamic therapy. Hence, we present a paradigm of perfect integration of DNA nanotechnology with nucleic acid amplification, thus paving a promising way to the improved performance of nucleic acid amplification techniques and their wider application.Nonenzymatic nucleic acid amplification techniques ( In order to clarify the important role of these biomarkers in the occurrence and development of disease and to achieve personalized therapy, increasing emphasis has been placed on rapid and sensitive imaging of the temporal and spatial distribution of biomarkers at the single-cell level. However, the intrinsic characteristics of most biomarkers, including low abundance and high structural similarity among family members, make their accurate detection challenging.Advances in genomics, proteomics and molecular pathology have generated many candidate biomarkers with potential clinical value.in vitro detection of low-abundance biomarkers with high sensitivity. These amplification methods require the involvement of multiple proteases. However, the delivery of proteases into live cells is currently difficult and may interfere with the expression levels of some biomarkers,et al. designed a type of localized molecular circuit on DNA origami for fast and efficient DNA computing, significantly accelerating the nonenzymatic amplification reactions.et al. developed a localized HCR on a DNA origami rectangle, in which the hairpins were successfully programmed to undergo a cascade HCR.et al. conducted DNA-based computing on a supramolecular polymer, and the kinetics of strand displacement and strand exchange reactions were accelerated 100-fold.et al. designed a proximity-induced DNA assembly for visualization of protein-specific glycosylation and protein dimerization.DNA-based biosensing and bioimaging studies have attracted more and more attention in recent decades.e.g. the HCR) to accelerate the reaction kinetics. By assembling DNA hairpins at the vertexes of quadrivalent tetrahedral DNA nanostructures (qTDNs), the reaction kinetics of the HCR is greatly accelerated due to the synergetic contributions of multiple reaction orientations, increased collision probability and enhanced local concentrations. Moreover, the participation of qTDNs can also efficiently improve the cell internalization efficiency and biostability of DNA probes. The qTDN-mediated HCR can solve the three major problems faced by the traditional HCR all at once. As a proof-of-concept, we demonstrated that it is a promising candidate for in situ imaging and photodynamic therapy (PDT).Inspired by this theory, we developed a tetrahedral DNA nanostructure-mediated nonenzymatic nucleic acid amplification reaction , H1 in qTDNH1 and H2 in qTDNH2 will be alternately opened, and qTDN-assembled DNA superstructures will be formed. Each qTDN contains four hairpin probes. These hairpin probes locate in different orientations, which might increase the collision probability between two hairpins from different qTDNs. In addition, the three-dimensional structure of the qTDN enables the qTDN-mediated HCR to be performed towards different orientations, which is beneficial for grabbing free reactants from the surrounding environment. What's more, once the hairpin at one qTDN vertex participates in the HCR reaction, the other three are naturally brought into the DNA superstructures, resulting in a great increase of the local concentrations of reactants. The synergy of increased collision chance, multiple reaction directions and increased local reactant concentrations will certainly be helpful to achieve accelerated HCR reaction kinetics.To accelerate the reaction kinetics of the HCR and promote its applications in live cells, we designed a qTDN-mediated HCR strategy . A TDN, Several characterization methods were used to demonstrate the feasibility of the proposed qTDN-mediated hyperbranched HCR. First of all, agarose gel electrophoresis (AGE) was used to verify the preparation of qTDNs and qTDNH1. As shown in Having demonstrated the feasibility of the qTDN-mediated hyperbranched HCR, we then verify its accelerated reaction kinetics compared to those of the traditional HCR using fluorescence assays. The traditional HCR can amplify a single molecular recognition event over one thousand-fold, thus making it a potential signal amplifier for detecting targets with low abundance. However, it has relatively slow kinetics inappropriate for rapid analysis. In addition, a prolonged reaction time may also increase the risk of undesired hybridization reactions.vice versa. Such an extraordinarily high FRET efficiency might make the qTDN-mediated hyperbranched HCR superior in biosensing and bioimaging applications.To achieve real-time monitoring of the HCR kinetics, we labeled h1 and H1 with the fluorescence donor Cy3 and h2 and H2 with the fluorescence acceptor Cy5, respectively. When the traditional and qTDN-mediated HCRs were successfully triggered by miR-21, the FRET between them shifted from an “off” to an “on” state. This signal output mode is superior to fluorophore/quencher systems, because false positive signals caused by probe degradation can be efficiently prevented.in vitro applications of the qTDN-mediated hyperbranched HCR and traditional HCR in target miR-21-sensing were compared. As shown in σ/S rule (σ is represents the standard deviation of the blank control (N = 10) and S is the slope of the calibration curve). Besides, such an excellent specificity of the hyperbranched HCR was achieved that even one-base mismatched target microRNA did not yield an observable FRET signal change, as compared with the blank control  can be obtained. In the above experiment, qTDN-assembled HCR reactants (qTDNH1 and qTDNH2) were used to demonstrate the feasibility of the qTDN-mediated hyperbranched HCR. Next, other valent TDN-assembled HCR reactants were prepared and used for TDN-mediated HCR studies. Their detailed compositions and integral structures are listed in Table S2 and Fig. S1a and b.To further elucidate the contribution of increased reaction orientations and local concentrations to the accelerated reaction kinetics of the HCR, different valent TDN-assembled hairpins were prepared to perform the TDN-mediated HCR. Since the TDN has four vertexes, a number of hairpins, ranging from one to four, can be easily modified on the TDN with varied valence. Herein, the valence of the TDN is defined as the number of CThen, the reaction kinetics of the different valence TDN-mediated HCR reaction was compared. By recording the Cy3 fluorescence decrease with the reaction time Fig. S2a, we founvice versa. Approximately 76% FRET efficiency achieved by the qTDN-mediated hyperbranched HCR is also the highest value for reported DNA-based FRET sensing systems.The FRET efficiencies of sensing systems were also greatly increased with a valence from one to three, and a comparable one resulted from tri- and quadri-valence Fig. S2b. This cavia macropinocytosis and caveolae-mediated endocytosis pathways. It is reasonable to infer that hairpins can be easily and efficiently delivered into cells with the help of TDNs. Moreover, owing to the protection of TDNs, both qTDN-assembled hairpins and the corresponding HCR products showed much better stability than unprotected ones in the traditional HCR. AGE analysis (e.g. qTDNH1) were still clearly observed within 4 h after incubation with 0.5 U mL–1 DNase I, a powerful endonuclease capable of effectively degrading both single- and double-stranded DNAs. Hyperbranched HCR products formed by the qTDN-mediated HCR remained stable even after 6 h. Fluorescence analysis showed that after treatment in a cell culture medium, the traditional HCR products formed by h1Cy3 and h2Cy5 exhibited a time-dependent decrease in the FRET signal after 4 h, but the FRET signal of HCR products formed by qTDNH1Cy3 and qTDNH2Cy5 remained almost unchanged within 36 h , we fouy3/h2Cy5 . In conty3/h2Cy5  and subse.g., Let-7a) was investigated. To achieve this, two fluorescent-labelled hairpins  for 4 h, obvious FRET signal output could be observed. When MCF-7 cells were transfected with a Let-7a mimic, a much higher FRET signal was observed compared to that of the untreated control. In contrast, Let-7a inhibitor-transfected cells gave a greatly decreased FRET signal output. All of these results suggested that the observed FRET signal was highly Let-7a expression-dependent, further suggesting that the proposed qTDN-mediated HCR method can be easily extended to the sensitive and reliable analysis of other target microRNAs in a biological matrix.To demonstrate the universality of the proposed strategy, its feasibility for the detection of another microRNA (e.g. doxorubicin (Dox) and methylene blue (MB)). To widen the application range of the qTDN-mediated hyperbranched HCR in live cells, we next combined the dual functions of qTDN-assembled hairpins in imaging and drug delivery, and investigated the feasibility of the qTDN-mediated hyperbranched HCR in imaging-guided tumor-targeted PDT applications. As a proof-of-concept example, we used qTDNs as the delivery carrier of MB, the photosensitizer commonly used in tumor PDT  generation ability after 650 nm irradiation, thus resulting in an irradiation time-dependent decrease in the absorbance of 1,3-diphenylisobenzofuran (DPBF), an extracellular 1O2 probe. In contrast, without irradiation, no obvious DPBF absorbance changes were observed  as the fluorescent probe of 1O2 (1O2 generation by qTDNHs@MB in cells. It is also demonstrated that qTDNs are appropriate carriers that efficiently deliver hydrophilic MB with poor cell/tissue penetration into live cells.The above experiments demonstrate that our proposed qTDN-mediated hyperbranched HCR works well for specific target-imaging detection in live cells, thus showing great potential in diagnostic applications. Because of their excellent stability, good cell internalization capacity and dense DNA nanostructure, TDNs are also promising carriers for the delivery of drugs that can intercalate into DNA duplexes (umor PDT .38,39 Wes Fig. S5. More thr qTDNH2 . After bobserved . In situe of 1O2 .40 HeLa Cy3 and qTDNH2Cy5, sensitive detection of miR-21 was still achieved, and the LOD (3.83 pM, Fig. S6c–eIn situ imaging of miR-21 in live cells provided a further demonstration. As shown in Cy3@MB + qTDNH2Cy5@MB. After further treatment of qTDNH1Cy3@MB + qTDNH2Cy5@MB in cells with 650 nm irradiation for 3 min, the cell viability sharply decreased to ∼20%. Collectively, these results demonstrated that our strategy can work well in “sense and treat” assays, thus suggesting promising applications in activatable theranostics of diseases.Because both the absorption and emission spectra of MB overlap with those of Cy5 Fig. S6b, we invein situ imaging of miR-21 inside live cells. Furthermore, stable qTDNs can also serve as good carriers for some drugs, and we demonstrated that the loading of the photosensitizer MB did not obviously affect their imaging diagnosis capability; therefore, constructing a theranostic platform to perform imaging-guided disease therapy is possible. Above all, we presented a facile way to improve the performance of the nonenzymatic HCR by utilizing multivalent qTDNs. With the rapid development of DNA nanotechnology, high-order, multifunctional DNA superstructures can conceivably be assembled by using other precisely designed DNA nanostructures with more valence states as building modules, such as polyhedra, bipyramids, cubes or prisms. They provide a potential target-triggered, in situ assembly tool for further exploration in nanotechnology and biomedical fields.In summary, we showed a new paradigm of a qTDN-mediated hyperbranched HCR strategy to accelerate the nonenzymatic HCR and promote its biological application in live cells. In this strategy, DNA hairpins, assembled at the vertexes of qTDNs, are used to perform the target-triggered hyperbranched HCR. The steric and multivalent characteristics of the qTDN can increase the collision probability of DNA hairpins by increasing the local concentrations and simultaneously initiating from multiple directions. The reaction rate of the qTDN-mediated hyperbranched HCR was about 70-fold faster than that of the traditional HCR. Due to the formation of hyperbranched HCR products, a stable FRET signal can be achieved within 20 min and up to 76% FRET efficiency could be obtained. The rapid reaction kinetics and ultrahigh FRET signal demonstrated its great potential in biosensing and bioimaging applications. In addition, the mediation of the qTDN can also promote the cell internalization of DNA hairpins, increase their biostability and thus facilitate the applications of the HCR in live cells. As a proof-of-concept, we demonstrated that the proposed qTDN-mediated hyperbranched HCR could be successfully applied in highly sensitive, specific and There are no conflicts to declare.Supplementary informationClick here for additional data file.


This study examines the benefit of using Ocean Mean Temperature (OMT) to aid in the prediction of the sign of Indian Summer Monsoon Rainfall (ISMR) anomalies. This is a statistical examination, rather than a process study. The thermal energy needed for maintaining and intensifying hurricanes and monsoons comes from the upper ocean, not just from the thin layer represented by sea surface temperature (SST) alone. Here, we show that the southwestern Indian OMT down to the depth of the 26 °C isotherm during January–March is a better qualitative predictor of the ISMR than SST. The success rate in predicting above- or below-average ISMR is 80% for OMT compared to 60% for SST. Other January–March mean climate indices  have less predictability  than OMT percentage deviation (PD) (80%). Thus, OMT PD in the southwestern Indian Ocean provides a better qualitative prediction of ISMR by the end of March and indicates whether the ISMR will be above or below the climatological mean value. Sea Surface Temperature (SST), representing the thin upper layer of the ocean, has been the main oceanographic parameter historically used to infer the influence of IOD and El Niño/Southern Oscillations, although the thermal energy required for the atmospheric processes comes from the upper ocean heat content (OHC). Many times, the thermal energy available in the upper ocean is not reflected in SST. For example, rapid heating and cooling events can quickly erase the thermal signature of subsurface warm or cold features4. Smith et al.5 observed that El Niño forecast models could be improved by initializing with the observed OHC. In addition, OHC has forecasting skill with more lead time as reported by Ji and Leetma6 and Latif et al.7. They found that forecasting skills improved, even with 6–12 months lead times, by initializing the numerical modelling forecast with the OHC. This predictability increases because interannual anomalies in OHC near the equatorial Pacific lead those in equatorial SST by several months8. This phase relationship is observed both in ocean models and in observations10. The atmosphere interacts with the upper ocean rather than with the surface alone, suggesting that ISMR predictions might be improved through the inclusion of upper ocean parameters. In this context, OHC is worthy of consideration for improving ISMR qualitative predictability .Predicting Indian Summer Monsoon Rainfall (ISMR) is a challenging problem. Although attempts are being made to improve the forecasting skill of the monsoon through numerical and statistical modelling, it remains difficult to predict. One reason for this low skill could be the incorrect representation of the ocean thermal energy that may lead to improper assessments of the influence of the oceans in the prediction models for extreme weather events. ISMR has a substantial impact on the Indian agriculture and economic growth. The Indian monsoon is a coupled phenomenon between ocean, land, and atmosphere. In addition to the atmospheric factors, oceans play a critical role in monsoon physics and prediction. The two main processes that affect ISMR are El Niño/Southern Oscillation13. Ali et al.15 suggested that OHC could be a better parameter than SST in predicting cyclone intensities in the Indian Ocean. Nagamani et al.16 reported that the OHC from the surface down to the depth of 26 °C isotherm (D26) in the Arabian Sea Mini Warm Pool (The region in the southeastern Arabian Sea from 4°–14°N to 68°−78°E) increased during 1998–2010. They speculated that this increase was responsible for the overall decreasing trend in the ISMR during this period. A widely used metric for the upper ocean heat energy is the tropical cyclone heat potential (used here as OHC), the integrated heat energy from the surface to D26, relative to the energy if that layer were uniformly 26 °C, with a minimum value of zero. However, since OHC cannot be integrated into purely atmospheric models, Ali et al.17 suggested converting OHC to Ocean Mean Temperature (OMT) of the surface to D26. They studied the relationships of ISMR with OMT and SST at each 2.5° box in the North Indian Ocean for different months and concluded that OMT in the southwestern Indian Ocean region , El Niño Southern Oscillation Modoki Index (EMI), and Indian Ocean Dipole Mode Index (DMI) are conventionally defined . The present study is useful because although there is typically modest variability in the monsoon season rainfall, small variations are very important for the agriculture and the economy of a country.In addition to interannual climate variations, OHC is useful for predicting tropical cyclone intensification22, the OHC and OMT, which depend on D26 in this case, have both spatial and temporal variations in this region. The D26 varies from 10 m to 100 m , the difference in rainfall with respect to the mean value, expressed as a percentage, without finding quantitative relation with rainfall anomaly. Nanjundiah www.imd.gov.in/section/nhac/dynamic/data.htm). This rainfall time series used 6955 rain gauges spread over India, including the hilly regions27 . The OHC field, the available heat energy in the upper layer, has dimensional inequality with SST and cannot be used in place of SST in the numerical models. To overcome this limitation, we converted the OHC fields to OMT fields using a few assumptions17. As the OHC from satellite and in situ derived observations has a good correlation having regression slope of close to one and a y-intercept of almost zero28, we used the following equation to compute OMT from OHC and D26 following Ali et al.17.p the specific heat capacity at constant pressure, T, the temperature (°C) of each layer of dz thickness, and D26, the depth of 26 °C isotherm. If we assume a mean temperature of the layer (from the surface to D26) as OMT, the above equation can be simplified as:We analyzed 25 years (1993–2017) of monthly rainfall data provided by the India Meteorological Department , the two parameters have the potential to be easily compared and replaced. OMT values thus obtained have data gaps at a few locations at 0.25 degrees spatial resolution. Hence, the monthly averaged OMT values have been obtained with a grid spacing of 0.5° × 0.5° using Cressman’sin situ temperature profiles. Since the temporal and spatial coverage of in situ observations is limited, we used satellite estimations providing more coverage in space and time. Nagamani et al.28 compared the satellite-derived OHC with the estimations from in situ hydrographic observations and reported a root-mean-square difference of 20.95 kJ cm−2, with a coefficient of determination, R2, of 0.65 and a bias of 11.27 kJ cm−2. Since we are using OMT in this study, we then further compared the OMT estimated from equation  and that estimated from the satellite-derived OHC has a scatter index, defined as root mean square error normalized to the in situ data mean, of 0.02 and coefficient of determination (R2) of >0.77 for both the North Indian Ocean, as a whole, and for the southwestern Indian Ocean, during January–March. Other statistical parameters for this comparison are given in Table The best and most reliable approach involves calculating OHC or OMT from equation  with thason Fig.  between 30 with a grid spacing of 1° × 1°, to compute the PD of January–March mean SST in the southwestern Indian Ocean. To show the importance of OMT compared to other oceanic indices, such as NINO3.4, EMI, and the DMI, these indices are obtained from the respective organizations as discussed below. Hadley monthly SST data30 was used to compute these parameters. We do not compute these indices separately; instead, we use them as they are for our comparison. The monthly NINO3.4 index, which is a central tropical Pacific SST based-index representing different phases of El Niño, is from Royal Netherlands Meteorological Institute climate explorer. The monthly EMI and DMI are from Japan Agency for Marine-Earth Science and Technology. DMI, representing the IOD phases and intensities during its life cycle, is defined as the gradient in SST anomaly between the western equatorial Indian Ocean (50°E–70°E and 10°S–10°N) and the southeastern equatorial Indian Ocean3 (90°E–110°E and 10°S–0°N). In addition, we computed another new index called ODMI, which is similar to DMI but using OMT in place of SST  and the southeastern equatorial Indian Ocean3 (90°E–110°E and 10°S–0°N). OHC obtained from satellite observations is up to the depth of 26 °C isotherm. To check if a shallower or a deeper layer would have been a better choice, we used Hadley temperature and salinity profiles (version: EN4) data31 to compute OHC of a few more depths. Complete details and description of the data/indices used in the present study can be found in Table We used monthly SST data from the Met Office Hadley Centre’s sea ice and SST data setFor an easy comparison between OMT, SST, and ISMR, the PDs of these three parameters are calculated. For example, the ISMR PD is calculated as the ratio of the deviation of a given year seasonal mean value from its June–September climatological mean value, divided by the climatological mean, and then multiplied by 100 as shown below:Spatial distributions of January–March climatic mean SST and OMT in the Indian Ocean during 1993–2017 are shown in Fig. 8, similar phase difference may also be present in the Indian Ocean. We further analyzed SST and OMT PDs in this study region to understand the predictability of this parameter for ISMR. For the purpose of easy visual comparison with ISMR PD, both SST and OMT PDs are multiplied by 10. A comparison of SST PD (red) and OMT PD (yellow) in the southwestern Indian Ocean with ISMR PD (green), shown in Fig. et al.16 also reported an overall decreasing trend in ISMR, which is attributed to the increasing OHC of the Arabian Sea Mini Warm Pool region. Furthermore, the lowest negative deviations in ISMR are −21.80% and −19.20% during 2009 and 2002, respectively, whose magnitudes are much larger than the highest positive deviations of 12.50% in 1994 and 5.7% in both 2007 and 2014. This indicates that the rainfall departures from the climatological mean during the years with below-average values of rainfall are greater overall than rainfall departures during years with above-average values. This is critically important, particularly for water resources development in the region, because the deficit rainfall in below-average years is not compensated by excess rainfall in above-average years even without consideration of the flood water going to the oceans. During the moderate below-average years , the ISMR PDs are −13.80%, −13.52% and −11.87%, respectively. These values are still more prominent than those during the above-average rainfall years. The maximum difference in ISMR PD between above- and below-average years is 34.30%, implying that the ISMR has high inter-annual variability, although normally the absolute value of this PD is much smaller than this extreme.We obtained the correlation coefficient for ISMR PD versus SST and OMT PDs separately for different months from January to May and for different 3-month combinations starting from January–March to March-May Table . OMT PD 32. Similarly, the OMT PD is successful in predicting the ISMR for 7 out of the 9 above-average rainfall years  below- (above-) average rainfall years  values in Fig. We carried out the following statistical analyses to assess whether OMT PD is a better ISMR predictor than SST PD:A 1000-sample permutations test revealed that the success rate for OMT PD is significant at the 1% level and that for SST PD it is significant at the 10% level. The difference between the success rates is significant at the 10% level.The equitable threat score accounts for the likelihood of random successes, with values > 0 indicating some skill, and a score of 1 indicating a perfect skill. We used separate equitable threat score to examine the effectiveness of determining the positive and negative signs. For SST, the equitable threat score is 0.020 for positive anomalies, and 0.094 for negative anomalies. Thus, SST has a little skill, but not much. In contrast, OMT has an equitable threat score of 0.295 and 0.405 respectively for positive and negative anomalies respectively. These differences are in part due to the probability of a false detection for an OMT-based estimate being about half that of an SST-based estimate.To test whether OMT PD is a confounder  or not, first the coefficient of SST PD with ISMR is obtained in a linear regression (ISMR = a0 + b1* SST PD). Then OMT PD is added to the regression (ISMR = a0 + b1* SST PD + b2* OMT PD). The coefficient of SST (b1) has significantly changed by adding OMT PD, indicating that OMT PD is a confounder.A standardized beta coefficient compares the strength of the effect of each individual independent variable to the dependent variable. The higher the absolute value of the beta coefficient, the stronger the effect. Standardized beta coefficients are the coefficients we get if the variables in the regression were all converted to z-scores before running the analysis. The multiple regression analysis of ISMR with OMT and SST PDs has the standardized regression coefficients of 0.92 (significant at 3% level for OMT PD and −0.71 (significant at 8% level) for SST PD. The student T-test T values for these two parameters are 2.43 and −1.86 respectively. These two results show that OMT is better correlated to ISMR than SST.In addition, the DMI and ODMI were compared with ISMR PD in Fig. 33 reported that the relationship between warm water volume in the tropical Pacific and El Niño Southern Oscillation has a much stronger lead time than the El Niño Southern Oscillation-SST relationship in predicting ISMR. Lopez et al.34 related the variability of the heat content/transport in the South Atlantic ocean and variability of monsoon at global scales. Zhu et al.35 studied the seasonality between the thermocline depth and SST relationship in the eastern equatorial Pacific. They observed that the thermocline-SST relationship is clearly weaker in spring (with deeper thermocline) than that in other seasons. The cause, according to them, is even though OHC (or the thermocline depth) has the strongest persistence during spring, its variations may not be communicated to the surface (represented by SST). Thus, the link between OHC and SST is depth dependent. This, probably, would not have been the case if OMT were considered.Among the six parameters studied, OMT PD averaged over January–March, performed better with two months lead time for a prediction. This statement is strongly supported by the differences in equitable threat scores, a 1000-sample permutation test, confounder analysis and comparison of standard regression coefficients indicating much more skill in the OMT-based forecast. Hence, OMT could be a better parameter as compared to SST, DMI, ODMI or EMI. This may be due to the fact that OMT better represents the upper ocean thermal energy conditions whose variations are mainly responsible for ISMR activity, whereas SST accounts only for the temperature of a very thin layer influenced by meteorological factors such as strong winds, evaporation, or thick clouds. In addition, compared to SST, OMT variations are more stable and consistent with less spread. Rajeevan and McPhadenAn important point worth discussing is the use of the mean temperature of the ocean layer down to the depth of 26 °C isotherm (the depth used for OHC computations). Although the use of OMT of this layer makes sense for TC studies as TC-induced mixing and cooling are weaker for higher values of OMT or OHC, such a reduced cooling during the pre-monsoon season cannot be ruled out. It could be related to vertical mixing. When OMT is high, mixing during the winter and spring does not cool SST as much, leading to warmer southwestern Indian Ocean waters, more moisture in the atmosphere, more moisture advected to the continent, and higher monsoon rainfall. Furthermore, there could be a remote oceanic influence associated with changes in OMT that SST does not capture. Westward-propagating Rossby waves could appear in OMT but not in SST, and then affect SST at the western boundary. The ocean thermal energy stored in the upper ocean represented by OMT in the southwestern Indian Ocean in January–March is generally a key parameter for the genesis and development of the following southwest monsoons. The variations of the stored ocean energy in different January–March may control the strength of the following ISMR by providing energy to the overlying atmosphere. This induces anomalous atmospheric circulation and moisture availability in the western Indian Ocean, including the Arabian Sea, a region where wind strength, direction, and moisture availability can significantly control the strength of ISMR. Thus, the use of SST is likely to provide poorer predictability of the ISMR relative to the use of OMT. These issues need to be addressed in a separate study, specifically making use of numerical models.In this study, we compared only the positive/negative ISMR years with respect to the climatological mean of 887.5 mm. We did not categorize rainfall as the deficit, below normal, normal, above normal, and excess years as the India Meteorological Department does. We also did not attempt to quantitatively predict or compare the ISMR PD with OMT PD. The aim of the present paper is to show that OMT could be a better parameter than SST and other climate indices for monsoon prediction. One possible way to make quantitative predictions could be to replace SST with OMT in numerical weather prediction models, which, however, is beyond the scope of the present study.26 is 35–85 m with 21 (84%) observations varying between 40 m and 70 m. We computed OMT PDs at seven other depths of the surface (5 m) to 15.1 m, 25.2 m, 35.3 m, 45.4 m, 55.7 m, 100 m and 150 m. The number of years mismatching with ISMR PD varies from 11 (correlation: 0.11) with OMT PD of the surface to 100 m to 18 matching or seven years mismatching (correlation: 0.23) with OMT PD of the surface to 15.1 m. Thus, we conclude that using OHC down to D26 to compute OMT PD is a better choice, possibly because this depth varies depending upon the atmospheric factors.To address if a much shallower or deeper layer than the depth of 26 °C would have been a better choice, we compute OMT PDs in southwestern Indian Ocean region for different layers using Hadley interpolated monthly mean temperature profiles with 1° spatial resolution. These temperature profiles are at 42 depth levels from 5 m to 5250 m. Out of the 25 years studied, the range of the annual mean DHere, we demonstrated statistical evidence that OMT is a better predictive parameter for the sign of ISMR compared to SST, DMI, ODMI, EMI and Nino3.4. The success rate for OMT in predicting the sign of the ISMR anomaly is 80% and significant at greater than 1%, far better than the other parameters considered.26 isotherm layer, has been recognized as an important parameter in cyclone forecasting, we extended the scope of this parameter to predict whether ISMR is above- or below-average value. We converted OHC to OMT so that the two parameters (SST and OMT) have the potential to be easily compared and replaced. Comparison of the correlation coefficient for ISMR PD versus SST and OMT PDs separately for different months from January to March and for 3-month averages from January-March to March-May revealed that OMT PD correlates better with ISMR PD compared to SST PD. Further, the number of mismatching years is least if OMT PD of January-March is considered. OMT PD is showing better performance than SST PD because of the following reasons: (1) The rapid heating or cooling events can quickly eradicate the thermal signatures of subsurface warm or cold features with which the atmosphere interacts rather than with the top thin layer of the ocean represented by SST alone and (2) SST variations that have smaller time scales introduce more noise and cause lower correlations with ISMR compared to OMT variations, which are more stable and consistent with lower spread. In addition, we also compared the OMT PD performance with ODMI another new index, which is similar to DMI but using OMT in place of SST, and other conventionally used climate indices such as NINO3.4, EMI, and DMI.Since the OHC, representing the mean temperature of the surface to D26 is a better choice, which could be because of the influence of the atmospheric factors on D26. The physical evidence for OMT in the southwestern Indian Ocean having a higher predictive value, and its ability to provide an improved quantitative prediction, need to be explored using numerical/dynamical models; here we present only the statistical evidence.Equitable threat scores, confounder analysis, standardized regression analysis and a 1000-sample permutation test suggest that among the six parameters/indices used, the January–March mean OMT PD in the southwestern Indian Ocean has the best relationship with ISMR PD. Since OMT PD of January–March has a better predictive value, by the end of March, we expect to predict qualitatively whether the monsoon would be above or below its climatological mean . Similar to other indices or parameters such as DMI, or El Niño/NINO3.4 or EMI, OMT PD helps in a qualitative prediction of ISMR — whether the monsoon is above or below the long period average. Comparison of this parameter estimated for other depths revealed that OMT PD of DAlthough we have already provided some reasons why OMT failed to predict ISMR in some years, the failure of predicting ISMR using OMT in some years implies the complexity of monsoon system determining the vagaries of ISMR, which are not always controlled by the ocean energy alone represented by OMT in the southwestern Indian Ocean. For example, the influence from other ocean basins associated with ENSO, atmospheric processes related to Madden-Julian Oscillation, Tropical Intraseasonal Oscillation in the time scales of 10–20 days, and the impact from the extra-tropic regions, which are at least partly independent of OMT in Indian Ocean, may also be important contributors to the variability of ISMR in both time and space.The OMT PD during January–March 2018 is −14.32, which may lead to below average rainfall during June–September 2018 with 80% probability.Supplementary Material


SBEs) in tetraploid potatoes could generate tuber starches with a range of distinct properties. Constructs containing the Cas9 gene and sgRNAs targeting SBE1,SBE2 or both genes were introduced by Agrobacterium‐mediated transformation or by PEG‐mediated delivery into protoplasts. Outcomes included lines with mutations in all or only some of the homoeoalleles of SBE genes and lines in which homoeoalleles carried several different mutations. DNA delivery into protoplasts resulted in mutants with no detectable Cas9 gene, suggesting the absence of foreign DNA. Selected mutants with starch granule abnormalities had reductions in tuber SBE1 and/or SBE2 protein that were broadly in line with expectations from genotype analysis. Strong reduction in both SBE isoforms created an extreme starch phenotype, as reported previously for low‐SBE potato tubers. HPLC‐SEC and 1H NMR revealed a decrease in short amylopectin chains, an increase in long chains and a large reduction in branching frequency relative to wild‐type starch. Mutants with strong reductions in SBE2 protein alone had near‐normal amylopectin chain‐length distributions and only small reductions in branching frequency. However, starch granule initiation was enormously increased: cells contained many granules of <4 μm and granules with multiple hila. Thus, large reductions in both SBEs reduce amylopectin branching during granule growth, whereas reduction in SBE2 alone primarily affects numbers of starch granule initiations. Our results demonstrate that Cas9‐mediated mutagenesis of SBE genes has the potential to generate new, potentially valuable starch properties without integration of foreign DNA into the genome.We investigated whether Cas9‐mediated mutagenesis of starch‐branching enzymes ( Solanum tuberosum L.) tubers has been the target of multiple types of genetic modification, for a variety of purposes. Earlier modifications were aimed at modification of starch properties for direct industrial use  of starch, amylopectin, consists of α‐1,4 linked glucoses with relatively frequent, clustered branch points formed by α‐1,6 linkages, amylose is a largely linear α‐1,4 linked glucose polymer. The amylose content of starch profoundly influences its functional properties  that eliminates a major form of SBE in the endosperm , which introduce α‐1,6‐linkages into starch. A naturally occurring high‐amylose, digestion‐resistant starch in maize arises because of a mutation  and SBE2 (designated SBE2.1 and SBE2.2), likely to be homoeologues , exon 5 (sgRNA2) and exon 6 , exon 8 (sgRNA6), exon 12 (sgRNA7), exon 13 (sgRNA8) and exon 14  extracted from leaves. In the first instance, mutations were identified by a reduction in amplicon size observed by agarose gel electrophoresis, indicating deletions between two sgRNA targets . We conducted detailed genotyping of the SBE genes and analysed the SBE protein content of these lines. None of the lines in which SBE1 alone was targeted had altered starch granules.Starch was prepared from tubers of senescing glasshouse‐grown plants propagated from plantlets with mutations in SBE1 and SBE2 mutated) was obtained by protoplast‐mediated transformation with construct 237  deletion of the DNA between two sgRNAs  was obtained by Agrobacterium‐mediated transformation with construct 230  were obtained by Agrobacterium‐mediated transformation with constructs 227 and 212, respectively  had extreme starch granule abnormalities, similar to those described previously for potato plants with strongly reduced SBE activity/protein  in 237/4‐44 starch was half that in wild‐type starch , 227‐25 and 212‐36 (SBE1 and SBE2 mutated) had different defects from those in the extreme mutant. Some granules in these lines were normal in appearance, but a large fraction was not. Starch from these lines resembled that of the extreme line with respect to the occurrence of nodular granules and granules with multiple hila, but there were no cracks and fissures associated with hila . They showed that—depending on the experimental conditions—up to 67% of plants recovered for a given sgRNA were mutated in multiple alleles, but only a very small percentage was mutated in all four alleles. Second, it is possible that some of our plants were genetic chimeras, containing both mutated and wild‐type cells. At least some of the calli that produced mutated shoots were chimeras: they gave rise to both mutated and unmutated plantlets, and—for calli regenerated from protoplasts—plantlets that had or did not have the Cas9 gene. For other lines, both sequencing data and the presence of abnormal starch granules in all tuber cells examined suggest that the genotypes were uniform and stable across the plant.Our data suggest two possibilities for the presence of both wild‐type and mutated n et al.  providedSBE1 and one SBE2 allele contained only small in‐frame deletions, resulting as expected in some residual SBE1 and SBE2 proteins. In other lines, there were no wild‐type alleles and no protein. It is not known whether homoeoalleles of SBE genes in potato are equally expressed and make equal contributions to protein levels in the plant. If their contributions are not equal, the extent to which a given mutation affects protein levels may depend upon whether it is in a strongly expressed homoeoallele or a weakly expressed homoeoallele. Allelic variation in gene expression has been reported frequently for hybrids and polyploids in several species , one et al., et al., et al., in vivo.It is technically extremely difficult to determine the extent to which the mutations affected the capacity for starch branching in tuber cells, because the assayable activity of SBE1 and SBE2 does not reflect their relative importance in starch branching. Antisense‐mediated reduction in SBE1 reduced total SBE activity in tuber extracts by up to 98% without any effect on chain‐length profiles below dp70 or on iodine‐based measures of amylose content  in severe et al. . Starch granules from these intermediate lines did not have the cracks around the hilum seen in the extreme lines, but a high proportion appeared to have arisen from multiple hila. Polarized light microscopy revealed multiple Maltese crosses within granules with relatively normal shapes, and some granules had irregular nodular shapes apparently arising from the fusion of multiple small granules. Strikingly, individual tuber cells also contained huge numbers of tiny granules. These were not present in tubers of wild‐type plants or extreme mutant lines. The clustered distribution of the tiny granules in isolated cells suggests that they were present in only one or a few amyloplasts per cell.1H NMR) of amylopectin in tuber starch were not strongly affected in the intermediate lines. Based on these observations, we propose that major loss of SBE2 has relatively minor impacts on amylopectin synthesis at the surface of growing granules, but strongly affects the process of granule initiation. The presence of huge numbers of tiny granules, granules with multiple hila and nodular granules points to a loss of control over the granule initiation process. Rather than initiation of a single granule per amyloplast as in wild‐type tubers  . A candidate transcript (id PGSC0003DMC400017627) mapped to scaffold PGSC0003DMS000001367. The translated coding sequence of SBE2 . A third candidate SBE  was identified by tBLASTn analysis of the transcriptome and BLASTp analysis of the predicted proteins.The translated coding sequence of SBE1 and SBE2 from S. tuberosum cv. Desiree (Table http://www.quiagen.com) was used for first‐strand cDNA synthesis  from which SBE1 and SBE2 were amplified  using Phusion polymerase . PCR amplicons were cloned into PCR8/GW/TOPO TA (Invitrogen) according to the manufacturer's instructions. Eight clones of each gene were sequenced. Multiple sequence alignment  identified single nucleotide polymorphisms consistent with the presence of two homoeologues of each gene  were identified for Cas9‐induced mutagenesis of s Figure .et al. , mutating the instance of the BbsI recognition site ‘gaagac’ to ‘gtagac’, and cloned into pUAP1 (Addgene # 63674) to create a new Level 0 part. The Level 1 assemblies containing sgRNA expression cassettes were assembled into the Level M Position 1 acceptor  together with a kanamycin resistance cassette  and a Cas9 expression cassette either with  or without a YFP C‐terminal tag . Twelve plasmids with different combinations of U6 promoters and sgRNAs were constructed . Culture preparation, transformation and regeneration of potato plants were performed according to Chetty et al. . When regenerated shoots were ~10 cm, tissue was sampled for genotyping. Plants were transferred to potting mix after clonal propagation from axillary buds.Plasmid constructs were transformed into y et al. . Pathogeet al.  and 220 μL of PEG solution. As a control, 20 μL dH2O was used in place of plasmid DNA. Parallel transformations were performed using constructs encoding Cas9 with or without a C‐terminal YFP tag ‐mediated protoplast transformation and plant regeneration were performed according to Nicolia et al.  with ming Figure .et al., 5 protoplasts/mL. After 3 days, samples transfected with plasmids encoding a Cas9:YFP fusion protein were examined by fluorescence microscopy to calculate transformation efficiency . After 2 weeks, strongly growing calli were transferred to shoot induction medium  and subcultured every 2 weeks. Samples were taken for genotyping. Shoots from calli with putative mutations in SBE genes were excised and grown on solid rooting medium (MS30 containing 0.8% (w/v) agar) without antibiotics before transfer to potting mix.After transformation, protoplasts were resuspended in nutrient‐rich liquid medium E  SDS; incubated at 65 °C for 15 min; mixed with 250 μL phenol and the upper aqueous phase collected following centrifugation ; and extracted with an equal volume of chloroform. After centrifugation DNA was precipitated with 100 μL isopropanol, collected by centrifugation, washed in 1 mL 70% (v/v) ethanol and resuspended in 50 μL dH2O.Genomic DNA (gDNA) was purified from leaves using the DNeasy 96 plant kit (Qiagen) or from calli as follows. Samples of 20 mg were crushed in 250 μL 200 mAgrobacterium‐mediated and protoplast transformation, respectively. Primers specific for SBE1 or SBE2 were used to detect Cas9‐induced mutations at the target. Amplicons were analysed by Sanger sequencing to determine the exact genotype. Putative mutants were propagated in a glasshouse, and genotyping was repeated on mature plants. Plants regenerated from protoplasts which were mock‐transformed with water in place of DNA were used as negative controls.The presence/absence of the Cas9 gene was checked by PCR with Cas9‐specific primers Table  on plantAgrobacterium‐mediated transformation were grown in 50‐L bags. Tubers were harvested shortly before or during plant senescence. Samples for starch assays and immunoblotting were taken from freshly harvested tubers using a corkborer, immediately frozen in liquid nitrogen and stored at −70 °C.Regenerated plants were grown in soil in a glasshouse with 22 °C day, 16 °C night and natural daylight. Protoplast‐derived plants were grown to maturity in 10‐L pots; plants derived from et al. (http://www.beckman.com), according to the manufacturer's instructions.Starch was prepared from tubers (30–100 g) according to Edwards et al. . Starch et al. , 5 mm Na2S2O5 (pH 7.0) for 3 days. Cells were separated on a slide by gentle pressure with a coverslip. Starch granules were observed with bright field and polarized light microscopy, either unstained or after staining with appropriately diluted (between twofold and 10‐fold) Lugol's iodine (2%).For cell separation  Agrobacterium‐mediated transformation and (b) PEG‐mediated protoplast transformation.Figure S6 Examples of confirmation of Cas9 expression and mutation of SBE genes in protoplasts 3 days after PEG‐mediated protoplast transformation.Figure S7 Detection of Cas9 transgene in protoplast‐derived plants.Figure S8 Sequence analysis of SBE1 from line 237/4‐44.Figure S9 Sequence analysis of SBE2 from line 237/4‐44.Figure S10 Sequence analysis of SBE2 from line 230‐51a and b.Figure S11 Sequence analysis of SBE1 from line 227‐25.Figure S12 Sequence analysis of SBE1 from line 212‐36.Figure S13 Sequence analysis of SBE2 from line 212‐36.Figure S14 Sequence analysis of SBE2 from line 227‐25.Figure S15 Images of tuber starch granules from mutant and wild‐type lines.Figure S16 Starch contents of mature tubers.Table S1 Numbers of plants with mutations at the intended targets.Table S2 Primers used in this study.Table S3 sgRNA recognition sequences in SBE1 and SBE2.Appendix S1 Supplemental Experimental Procedures: 1H NMR and HPLC‐SEC.Click here for additional data file.


ObjectivesDue to the high demand for all-ceramic restorations, monolithic zirconia restorations are nowadays frequently used. With the demand for adult orthodontic treatments, orthodontists need to be mindful of the quality of their brackets bonding to this type of material, as it requires special conditioning. This study aimed to compare different surface treatments of zirconia when bonding metal or ceramic orthodontic brackets. The objectives are to compare the shear bond strength; the amount of adhesive remaining on the surface of the material; the incidence of adhesive, cohesive, and mixed failures; and the occurrence of zirconia fractures.Materials and MethodsForty monolithic blocks of zirconia of a diameter of 10 mm and a length of 10 mm were prepared and randomly divided into two groups (n= 20): metallic or ceramic brackets. Each group was subsequently divided into two subgroups (n= 10) depending on the surface preparation (laser treatment or airborne particle abrasion): SMB , SCB , LMB , and LCB . The samples were tested for shear bond strength using a universal testing machine. The adhesive remnant index and the occurrence of zirconia fractures and different types of failures were assessed by optical and electron microscopy.Statistical AnalysisResults were analyzed using analysis of variance.ResultsThe differences were significant between the metallic  and ceramic  bracket groups with regard to shear bond strength, with respectively 23.29 ± 5.34 MPa, 21.59 ± 4.03 MPa, 20.06 ± 4.05 MPa, and 17.55 ± 3.88 MPa. In terms of surface treatment, no statistical differences were found between the different groups.ConclusionMetal brackets have a greater bond strength than ceramic brackets when cemented to zirconia. The surface treatment of zirconia surface has no influence on the shear bond strength. Moreover, it presents a Young's modulus of 210 GPa, which is similar to that of stainless steel alloy.The aesthetic demands of patients are increasing, and all-ceramic fixed partial dentures (FPDs) meet their needs. Research has led to the development of zirconium oxide, or zirconia ZrO, a mateOrthodontists are regularly faced with patients who present monolithic zirconia restorations. But despite all its qualities,Laser treatments are constantly evolving, and are used in general practice, for certain treatments in periodontics, as well as in dental surgery, surgery, and other fields.Some studies have shown that ceramic brackets are recommended on surfaces like zirconia ahead of metal brackets,The aim and objective of this study are to evaluate the influence of orthodontic bracket material  and zirconia surface treatment (airborne particle abrasion or laser-etching) on the shear bond strength of these brackets to surface treated monolithic zirconia blocks. We also investigated the amount of residual cement on the blocks after failure by means of the adhesive remnant index (ARI) using an electron microscope and an optical microscope. We then observed the occurrence of adhesive or cohesive failures to determine whether the bonds created between the interfaces were stronger than the bonds within the materials themselves (or vice versa), as well as the occurrence of restoration material fractures.n= 20): metallic  and ceramic  , shade B2-B3, 10 mm in diameter and 10 mm in length, were prepared, and randomly divided into two groups  , and sun= 20), the surfaces were prepared by airborne particle abrasion using 25 μm aluminum oxide  for 20 seconds at 2.5 bar and a distance of 10 mm.For half of the samples (n= 20), the surfaces were covered with graphite powder (HB pencil) to increase its energy absorption and then subjected to erbium-doped yttrium aluminum garnet (Er:YAG) laser radiation . The laser was set at a wavelength of 2940 nm, pulse duration of 50μs (SSP), power of 2 W, pulse repetition rate of 10 Hz, and an energy density of 200 mJ. An R14 handpiece was used and equipped with a sapphire tip of a diameter of 0.8 mm. The air/water spray ratio was set at 4/4. The sapphire tip was held perpendicular to the surface of the block at an approximate distance of 0.5 mm. The surface of the zirconia block (78.54 mm2) was then subjected to radiation at a speed of around 2 mm/s for 10 seconds using horizontal scanning.For the other half  was applied to the adherend surface of the zirconia block for 20 seconds using an application brush, after which the entire adherend was suitably dried using a moderate, oil-free air spray.The primer  was measured into a mixing palette and applied to the surface of the conditioned zirconia surface in a thin, uniform film using a microbrush. Since the primer is photopolymerizable, intense exposure to ambient light was avoided and the intensity of the surgical lighting was reduced during application.2(–10%/+20%): light oriented either on the interproximal surfaces of the metallic brackets for 10s or perpendicularly to the ceramic brackets for 20 s.A sufficient quantity of bonding agent  was applied to the base surface of each bracket. As soon as the bonding agent had been applied, the bracket was lightly placed on the surface of the zirconia block, its position adjusted, and it was then firmly pressed down. Excess adhesive around the bracket base was delicately removed with a probe without moving the bracket. Lastly, we performed photopolymerization using a curing light  with an intensity of 1470mW/cmThe blocks were then placed in a cold-curing resin  except for the surface where the bracket had been cemented.The shear strength test was conducted using a universal testing machine . Shear stress was applied in a downwards direction parallel to the surface of the zirconia block at a speed of 0.5 mm/m .The load applied was recorded in N and shear strength in MPa. An optical microscope (magnification x20)  was used to determine the ARI score after failure.The ARI score is represented by a scale with four levels, from 0 to 3:0. No adhesive left on the surface1. Less than half of the adhesive left on the surface2. More than half of the adhesive left on the surface3. All of the adhesive left on the surface after failure.Using the optical (Leica) and electron  microscopes, we were able to check for adhesive, cohesive, and mixed failures as well as fractures of the zirconia restoration material. The steps in the protocol were all performed by the same operator.Statistical analysis was conducted using IBM SPSS Statistics, version 23 .The values obtained on bracket debonding during shear testing are described inp> 0.05), we accepted the null hypothesis that the variances were equal. The variances were therefore deemed to be similar.Descriptive statistics, including the mean, standard deviation, median, and minimum and maximum values for shear strength (MPa), were calculated. Because this test was not significant  was conducted to determine whether there existed a statistically significant difference between the groups. The differences were significant between the metal  and ceramic  bracket groups with regard to shear bond strength, with respectively 23.29 ± 5.34 MPa, 21.59 ± 4.03 MPa, 20.06 ± 4.05 MPa, and 17.55 3.88 MPa. The SMB group  showed the highest shear bond strength values, whereas the lowest was that of the LCB group (Er:YAG laser and ceramic bracket).p< 0.05) between the different types of bracket. Conversely, the groups treated with airborne particle abrasion or laser-etching did not display any statistically significant differences in terms of their shear strength.The ANOVA was significant . Thus, there was no statistically significant difference between the groups.Statistical analysis in the form of the Kruskal–Wallis test was conducted on the variable “ARI score” and was not significant . The aims are to evaluate the influence of brackets type and influence of zirconia surface treatment on the shear bond strength of orthodontic brackets to zirconia surfaces.2O3),It is difficult to achieve a long-term bond to ceramic surfaces. The findings of previous studies confirm that applying hydrofluoric acid to zirconia does not result in effective retention.2, which create micro-cracks.Laser is employed in multiple fields of dentistry,We did not observe any statistically significant difference in this study between the samples based on the surface treatment they were subjected to, although the highest mean shear strength was achieved by the airborne particle abrasion group.Some studies have shown that ceramic brackets are recommended for surfaces like zirconia ahead of metal brackets,In this study, the samples that were given metal brackets displayed greater shear strength, and a statistically significant difference was indeed found between the metal bracket group and ceramic bracket group. Our finding matches that of the study by Mehmeti et alHobson et alWhen debonding a bracket, it is important not to alter the structure of the enamel while ensuring that the residual adhesive on the surface of the tooth is minimal. This applies to ceramic materials as well. Our objective during debonding is to minimize cohesive damage to the zirconia and leave as little bonding agent as possible on the surface of the restoration. That is why a low ARI score is desirable, as well as avoiding any cohesive failure within the zirconia.The LMB group displayed the lowest mean ARI score, followed by the SMB, LCB, and SCB groups. The groups with ceramic brackets had higher mean ARI scores than those with metal brackets. However, after analysis, there was no statistically significant difference between these groups.There are two categories of failure when two indirectly bonded materials come apart:A cohesive failure occurs within the bonding agent, the bracket, or the zirconia and indicates that the bond in the interface is stronger than that within the material.An adhesive failure occurs at the bracket/cement or zirconia/cement interface and it indicates that the bond is weaker at the interface between the cement and the material (the zirconia or bracket).In this study, we found no pure adhesive failures or cohesive failures within the brackets or zirconia blocks, only mixed and cohesive failures within the resin. None of these failures damaged the zirconia or the brackets.Within the limits of this study, we can conclude that metal brackets have a greater bond strength than ceramic brackets when cemented to zirconia. No statistically significant difference in shear strength was uncovered between the surface treatments.As regards ARI score, the sample groups did not appear to have any statistically significant differences between them.Sibel Cetik: Data analysis, manuscript preparation/editing/review.Thaï Hoang Ha: Literature search, English translation, manuscript review.Léa Sitri: Experimental studiesHadrien Duterme: Data acquisition and analysis.Viet Pham: Date analysis and literature searchRamin Atash: Concept, design, protocol.All authors approved the final version of the article.


Decision process about the acceptance of the deceased donor kidney for transplantation might be challenging. Although histological evaluation of pretransplant donor kidney biopsy provides reliable information regarding cortical necrosis, vascular thrombosis, extensive global glomerulosclerosis, and interstitial fibrosis/tubular atrophy, only electron microscopy enables thorough and reliable insights into microvasculature changes of kidney graft. The aim of the present paper is to briefly present two cases of early kidney graft loss. In one case, the donor was exposed to long-term extracorporeal membrane oxygenation (ECMO); in the other case, the donor experienced Takotsubo cardiomyopathy. In both cases, light microscopy of pretransplant biopsy found no pathology or significant discrepancy in morphology of kidney graft, while electron microscopy revealed severe endothelial dysfunction of renal microvasculature. Our results suggest that severe injury of renal microvasculature with relatively preserved tubular epithelium may be associated with some conditions of deceased kidney donors leading to early kidney graft nonfunction and loss. Further studies are needed to determine prognostic significance of severe ultrastructural microvasculature lesions and to evaluate disease states and conditions that could be associated with severe endothelial dysfunction of kidney graft. Since 1950s, when the first renal transplantation was performed, continuous progress in the replacement therapy has resulted in improved kidney transplant outcomes. Long waiting lists for renal transplants and shortage of living and standard criteria deceased donors have led to the expansion of donor pool, involving an increasing number of suboptimal and marginal donors , 3. A feTo help judge the quality and to avoid unacceptably high discard rates of deceased donor organs, pretransplant kidney biopsy is recommended . In our According to our experience, both light and electron microscopies of pretransplant biopsy offer valuable information on graft quality. Light microscopy offers an insight into organ pathology, including acute cortical necrosis, acute tubular injury, arterial/ arteriolar thrombosis, and chronic lesions , while electron microscopy provides insights into the ultrastructural changes that cannot be appropriately evaluated by light microscopy, such as basement membranes, microvasculature endothelium , or cellular structures of renal epithelium. Such assessment  of a kidney graft enables precise evaluation of the quality of the kidney graft and accurate differentiation of the preexisting changes from those arising after transplantation. In addition, it enables critical evaluation of observed ultrastructural alterations of the kidney graft in cases of early graft loss.Recently, we were faced with two cases of early graft loss. Although histologic evaluations of pretransplant biopsies were promising in both cases, subsequent electron microscopy revealed severe endothelial dysfunction of renal microvasculature. It is tempting to speculate that the injury of renal microvasculature on the pretransplant renal biopsy might be associated with early kidney graft nonfunction and loss. Therefore, the aim of the present paper is to demonstrate our recent findings and to briefly discuss potential risk factors associated with endothelial dysfunction of exposed kidney grafts.Endothelial dysfunction, more appropriately considered as endothelial activation, represents a switch from a quiescent phenotype toward phenotype showing host defense response. Endothelial cells can lose integrity, progress to senescence, and detach into the circulation .On transmission electron microscopy, activated glomerular endothelial cells are swollen, with extensive microvillus transformation of endothelial cell membrane, and loss of fenestration. In severe injury, endothelial cells may rupture leading to loss of intracellular content/debris into capillary lumen followed by detaching and loss of endothelial cells .To get an insight into the endothelial cell alterations a blinded light and electron microscopy analysis was performed on 50 consecutive pretransplant biopsies obtained from deceased donors after brain death. Tubular injury was assessed according to preservation of tubular epithelial cells, preservation of apical brush border evaluated in periodic acid Schiff (PAS) stain, and estimated percent of vacuolization in tubular epithelial cells.By light microscopy, glomeruli in all biopsies appeared unremarkable -PAS. ChlInterestingly, ultrastructural examination showed similar features in 48/50 cases. Glomerular endothelial cells showed preserved fenestration with scattered endothelial projections (microvillus transformation of cell membrane). Focal mild endothelial cell swelling was observed in some cases with no or little intracellular debris in glomerular capillary lumens and no detached endothelial cells . EndotheIn contrast, 2/50 cases with increased intraglomerular neutrophil granulocytes showed significant and severe ultrastructural changes of renal microvasculature without significant changes in tubular epithelium Figures .Changes in glomerular endothelial cells looked similar in both cases Figures . There wInterestingly, tubular epithelium showed only minimal changes with slightly decreased brush border similar to other 48 cases (changes very likely related to cold ischemia). Desmosomes in tubular epithelium were preserved Figures . NeverthIn the first case Figures  and 3, tIn the second case Figures  and 4 thTransplant kidney biopsy performed 4 days after transplantation revealed glomerular capillary thrombosis and arterial fibrinoid necrosis consistent with acute glomerular and vascular thrombotic microangiopathy, changes not observed in the pretransplant biopsy. Repeated donor specific antibody measurements were negative and there were no other histologic characteristics of antibody-mediated rejection. It was supposed that recipient could have recurrent aHUS, which was confirmed by recipient's complement alternative pathway dysregulation .The time range of cold ischemia storage in 48/50 grafts was 7h21 min-29h25 min, in deceased donor exposed to ECMO the time of cold ischemia was 16h22 min, and in deceased donor exposed to Takotsubo cardiomyopathy it was 9h19 min.It is known that endothelial dysfunction/activation can be associated with numerous factors, for instance, risk factors originated in the donors , transplantation procedures (ischemia-reperfusion), or associated with the recipient (immunologic and/or pathophysiologic (miss)match) . However»traditional« risk factors. Discussion on these topics could, however, be extremely broad and exceed the range of this article. Therefore, we would like to stress out and briefly discuss only factors that clearly stood out from other 48 cases and may be importantly associated with the graft outcome. According to the history of the donors two outstanding clinical peculiarities are noticed: in the first case exposure of the donor to ECMO after massive myocardial infarction with successful resuscitation and in the second case donor's experience with Takotsubo cardiomyopathy.Therefore, extremely severe and discrepant endothelial injury observed in the two above described kidney graft donors was very unlikely associated with α and IL-8, [Extracorporeal membrane oxygenation (ECMO) is used as a standard therapy in critical care for patients with cardiorespiratory failure and nowadays also to manage potential organ donors following cardiopulmonary failure , 14. It nd IL-8,  which alnd IL-8, . It was nd IL-8, .The outcomes of DBD kidney transplants procured from donors on ECMO have been shown to be comparable to those from non-ECMO donors . HoweverTakotsubo stress cardiomyopathy occurs in the setting of a severe mental or physical stressor, predominantly affecting postmenopausal females, and in the absence of obstructive coronary artery disease. Typically, there is transient reduction in left ventricular systolic function with specific regional wall motion abnormalities . AlthougThe results show that while myocardial function has already recovered after acute insult, endothelial cells are more severely impaired than smooth muscle cells, and that this injury persists beyond myocardial stunning. Thus, endothelial-dependent dysfunction can still impair vasodilatation, while ventricular dysfunction is successfully mechanically supported or has already resolved . This foOur observations show that severe endothelial injury of kidney graft microvasculature seen only on ultrastructural level may occur before any significant injury is observed on pathohistological level in preimplantation kidney graft biopsies. These extreme injuries to microvasculature, which are associated with early graft failure, could be promoted by some specific conditions in donor and/or recipient such as long-term ECMO employment, postresuscitation myocardial stunning, or Takotsubo cardiomyopathy. Further studies are needed to determine prognostic significance of severe ultrastructural microvasculature lesions and to evaluate other disease states and conditions that could be associated with severe endothelial dysfunction of kidney allograft.


Academic journalism is a competitive and complex realm. It is a world of unrelenting struggle for recognition, reputability and acceptance where promoted research is constantly exposed to criticism over quality and relevance to bolster journal metrics.2There are three main aspects of scholarly metrics: impact, speed and reach. Impact factors are based on the number of journal citations, usually over a 2-year period. Speed is related to review turnaround time and time to publication. Reach pertains to global authorship and readership.Indexing on the other hand reflects on the journal’s scientific quality. It places the journal on a database, allowing increased visibility, accessibility and readership, and ultimately reputability as a respectable and reliable source in the field of research. The prestige associated with indexing holds relevance for several reasons. Firstly, several academic institutions only recognise indexed publications, particularly for teaching and medical colleagues. Thus, an indexed journal is likely to receive more manuscripts than a non-indexed journal. Secondly, not all indexers are considered equal, despite the availability of several established, well-known databases.3 As such, indexing services worldwide have strict inclusion criteria and stringent evaluations prior to acceptance. The difficulty faced by authors and journals is deciding on which indexers to aim for.4 These findings affirm a positive research outlook for South Africa and South African journalism, despite the challenges we face.Based on the Report on the Evaluation of the 2017 Universities’ Research Output released in March 2019, there has been a steady increase in research productivity across all local universities over the 11-year period evaluated. This is attributed, in part, to institutional strategies and policies that encourage global exposure of local research. The report also documents significant publication growth in South African journals.Imaging-South Africa/Suid Afrika in 1990. This was subsequently named the SA Journal of Radiology (SAJR) in 1996 and is currently available as an open access digital journal, running its 23rd volume. The SAJR is indexed with Scopus (managed by Elsevier); SciELO SA; Web of Science Other Coverage, Emerging Sources Citation Index (ESCI); Directory of Open Access Journals (DOAJ); EBSCO Host; GALE, CENGAGE Learning; Google Scholar; Hinari; ProQuest; African Index Medicus; Norwegian Register for Scientific Journals, Level 1; and has most recently been accepted for inclusion in PubMed Central . This broad exposure places the journal at an advantage to evolve on multiple platforms. The journal’s new association with the esteemed and established PubMed Central is a significant milestone for carving the future of the SAJR in more ways than ever before.Supported by the Radiological Society of South Africa, local radiology publishing was first commenced as SAJR, to all those who fostered belief and pride in the journal and to all those who pledged to sustain the journal. Today we are proud to have a radiological journal that continues to reach new heights with a promising future ahead. I stand firm in my goal as the editor to promote the journal and share our research with the world.Thank you to all those who have engaged with the ‘If we knew what we were doing it would not be called research, would it?’ – Albert Einstein