We present a protocol for the design, fabrication and characterisation of laser-induced ultrasound transmitters with a specific, user-defined frequency response for the purpose of ultrasound tomography of large-volume biomedical samples. Using an analytic solution to the photoacoustic equation and measurements of the optical and acoustic properties of the materials used in the transmitters, we arrive at a required mixture of carbon black and polydimethylsiloxane to achieve the desired frequency response. After an in-depth explanation of the fabrication and characterisation approaches we show the performance of the fabricated transmitter, which has a centre frequency of 0.9 MHz, 200% bandwidth and 45.8 In one such example an optical fibre was fitted with a selectively absorbing film for LIUS excitation and an integrated optical sensor based on a ring resonator, to allow miniaturised PA/LIUS sensing and imaging Most of the applications of LIUS in biomedical imaging from the literature share the characteristic that they tend to focus on imaging relatively small targets like zebrafish and mice, or intravascular imaging. In these cases it is advantageous to use LIUS sources with high optical absorption, to both maximise the generated peak pressure and bandwidth of the generated acoustic pulses to improve spatial resolution. Recent work by Joo et al. The current work presents a detailed look at the design, fabrication and characterisation of unfocused LIUS transmitters for large-volume ultrasound CT of, for example, the human breast. The use of LIUS can be beneficial for the prevention of crosstalk, because the transmitters are intended to be used with a high density of highly sensitive PZT detectors. Further, this decoupling of transmission and detection allows for the independent optimisation of both. Since it is important, in this case, to detect the LIUS signal with as many detectors as possible per shot, a large opening angle in addition to a lower centre frequency for transmission through around 10 cm of tissue are desirable features. We present the design considerations in terms of geometry and dimensions and an analytic approach to estimating the output characteristics of such a transmitter. A protocol for fabricating such a transmitter will be presented in detail. We also show the methods used to optically and acoustically characterise the materials in the active element and the response of the finalised transmitters. While the transmitters presented here are designed for a specific imaging application and have the properties to match, the design and fabrication approach can be easily adapted to attain any desired set of transmitter properties using low-cost, readily available materials.22.1The transmitters are designed to match with a reasonable precision to the response and sensitivity of a custom detector . The detector frequency response peaks at 1 MHz with a bandwidth of roughly 100%. The remaining pressure after transmission across the imaging volume, a distance of some 30 cm, needs to be above the minimum detectable pressure, around 0.5 Pa. An opening angle of at least 30For this particular transmitter design the physical dimensions are restricted by the need to use them with an existing set up 2.22.3The properties of the generated acoustic pulse can be influenced, within our design, by the optical absorption coefficient (2)The first term is due to the direct pulse from the initial pressure distribution while the second term denotes its reflection off the back surface of the absorber, with The pressure detected at a given point as a function of time can be described as follows, by solving the photoacoustic equation with the Green’s function approach for an optical delta pulse (7)The two ranges of In the following we assume the laser spot size to be significantly smaller than the diameter of the absorber, which is thus treated as though it extends infinitely in the lateral dimension (11)Where the following constants and variables are defined, for compactness’ sake: Evaluating Eqs. 2.42.4.1The base material for the LIUS transmitters is polydimethylsiloxane , which is mixed with an appropriate amount of carbon black  for optical absorption, 45 g of PDMS with 3.63 g of CB for a weight percentage of 8.07%. The transmitter casings are made of machined polymethylmethacrylate (PMMA), and the backing consists of a disc-shaped borosilicate glass microscopy cover slip with a 5 mm diameter and 2.4.2Next, in a separate container or dish, a desired amount of stock mixture is weighed, denoted After adding the required weight of clear PDMS a disposable plastic pipette is used to add curing agent of the desired 10% of the total PDMS weight, after which it must be well mixed with a small spatula until it appears homogeneous, as the volume is too small to use a mechanical mixer. To facilitate the production of multiple transmitters the mixture is cured in a slab by transferring it to a flat-bottomed rectangular container, degassing in a vacuum vessel at −1 bar and regulating the thickness with a PMMA plate on a translation stage. Any excess liquid will flow past the edge of the plate and is cut off after curing. The mould is then placed in an oven at 70 °C for 90 min to cure.Once cured and cooled, 10 mm diameter discs are cut from the slab with a mechanical hole punch and a hammer. A small drop of cyanoacrylate adhesive is deposited on one face of a cut disc and smeared out into a thin layer with a needle tip. A glass backing disc is gently but firmly pressed onto the glue for tight adhesion to the disc. The tube segment and fibre mount need to be bonded coaxially, for which purpose a cylindrical alignment piece was machined, shown in image III on the right-hand side of 2.5Before fabrication of the transmitters could begin, the dependence of the optical properties of the CB/PDMS mixture in the concentration of CB had to be determined. Once known, the acoustic properties of a sample with the required optical properties also had to be characterised. This section will describe the various characterisation approaches taken.2.5.1d is the particle diameter and −20 mm−1 for the CB concentrations in the range of interest, many orders of magnitude below The required weight concentration of CB is determined by measuring the optical transmission spectrum for a range of samples of PDMS with CB contents between 0.03% and 0.28%, beyond which the transmitted light is insufficient for an accurate measurement. Any higher concentrations could not be measured by the spectrophotometer . The samples are prepared by mixing a range of concentrations from the previously prepared stock solution and placing a drop inside a circular, −1] is the speed of sound in water.Once the proper CB-percentage for the desired photoacoustic response is known, a precursor slab for a desired number of transmitters is fabricated according to the description in Section While the acoustic attenuation between roughly 0 and 4 MHz can be determined from transmission measurements on this block, it was deemed preferable to measure it over a broader frequency range. For this purpose a pair of disc-shaped samples of 2.5 cm diameter and 3.97 and 5.97 mm thicknesses was also cast. In this manner the sound speed can be measured with greater accuracy due to the smaller relative uncertainty in the measured sample thickness, while the attenuation will be known over a broader frequency range. The acoustic attenuation in dB per cm as a function of frequency can be determined from b are known they can then be used to estimate the impact of acoustic attenuation on a transmitter of a given thickness.Once the values of 2.5.2A fibre-optic needle hydrophone system  is used to characterise the response of the transmitter under illumination by a 3.6 mm diameter (1/e) Gaussian spot from a compact diode-pumped pulsed Nd:YAG laser  emitting 4.7 ns pulses at a 100 Hz repetition rate at a 1064 nm wavelength. The light from the laser is coupled into a multimode optical fibre , in a fibre multiplexer described in more detail in previous work 33.1−1 and 10 mm−1 at a depth of 300 mm with an optical pulse energy of 1 mJ and −2 gives the signals and spectra shown the insets in −1, with a bandwidth from 0 to 5.5 MHz and peak-to-peak pressure of 49.3 Pa at 300 mm in water. The signal and frequency spectrum for −1 are shown in −1.Evaluating Eqs. 3.23.2.1−1 requires a CB concentration of 0.075% by weight. Calculating the expected value of the scattering coefficient −1, 20 orders of magnitude below 3.2.23.3−2, at a distance of 6.2 mm from the transmitter surface. An overview is shown in −2), exhibiting oscillations in the rising flank of the negative peak currently suspected to indicate detachment of the backing from the absorbing layer. There appear to be no oscillations visible in the pre-damage curve in The generated peak-to-peak pressure was measured using illumination pulse energies between 809 −2 should allow for damage-free operation.The detachment may well have worsened at higher pulse energies, partially explaining the decrease in generated pressure. At higher pulse energies the amplitude of the internal-reflection peak at −2. For further measurements, using an undamaged transmitter, a liberal safety margin to prevent damage was maintained. The transmitter was illuminated with pulse energies of 950 −1 e−1 points at each depth, from which the opening angle of 45.83.4The LIUS transmitters presented in this work are still very much prototypes. Were they to be made for wider distribution and application some further improvements would be advised. The glass backing is quite fragile in the current design, meaning even fairly light mechanical pressure applied to the front face of the transmitter can lead it to break or detach. This immediately deteriorates the signal quality, if not necessarily the pressure, in unpredictable ways. One way to make it more robust would be to have the backing not simply attached to the PDMS, but integrated with the casing. This could be done by using a wider glass disc or machining the PMMA casing to incorporate a thin layer of material on top of the air pocket. To lower the chances of the backing detaching a different bonding mechanism may also be considered. In microfluidics highly robust bonding of PDMS is regularly performed to both glass, through oxygen plasma treatment While PMMA is a low-cost material that is also easy to machine, a further simplification of the fabrication process might be achieved by 3D-printing the casings instead. As long as the precision is high enough to allow for the placement of the optical fibre with variations of no more than As described above, the current method of fabrication includes a considerable potential for variability in transmitter properties, due to the hand-made nature of the transmitters. In applications using multiple transmitters in concert, it is therefore important to characterise each transmitter well, and to be sure that their properties do not change during an measurement. Even if there is an appreciable variation in transmitter pressure output, or even centre frequency, knowing exactly what these variations are will let them be taken into account. Ideally, in future improvements to the fabrication process any variability would be minimised by more standardised and automated fabrication steps, as well as by ensuring robust bonding methods that do not vary with time and are not susceptible to optical damage in the course of normal operation.The calibrated fibre-optic hydrophone used for the characterisation measurements was well-suited to determining the field geometry, the active sensor having a diameter of For integration into a hemispherical large-volume PAT/USCT system the positioning of the transmitters needs to be considered carefully. Depending on the requirements of the PAT illumination and the density of detecting elements, a minimum number of LIUS elements for sufficient-quality images should be arrived at. Considering the desire to insonify as much of the imaging target as possible with each LIUS shot, it is likely that a narrow ring nearer the rim of the bowl would be optimal. In this manner a large range of angular positions could be covered by perhaps a few tens of transmitters. With such a number of transmitters in mind, the illumination method should also be addressed. One method to efficiently illuminate such an array of LIUS transmitters sequentially could be through the use of a fibre-optic multiplexer based on a matrix of fibre inputs, selectively illuminated by a laser via galvanometer mirrors and a scanning lens, as we have presented in previous work A final consideration for the integration of the LIUS transmitter into a multimodal system with a PAT capability is the generation of parasitic signals. When illuminating the imaging volume for PAT measurements, light hitting the front face of the LIUS transmitters is absorbed and can generate unwanted signals orders of magnitude stronger than the actual PA signals. This is highly undesirable and has to be addressed by preventing optical absorption in the front face of the LIUS transmitters. One of the easiest ways to do this, with minimal impact on the LIUS signal quality, would be to place gold foil stickers on the front faces of the transmitters, which would reflect most of the light and drastically, if not completely, reduce parasitic signals. A similar approach has already been shown to work in preventing parasitic signals from optically absorbing ultrasound detectors in a photoacoustic mammoscope For future work, several options present themselves to further improve control over the output of the LIUS transmitters. For one, the addition of a broadband acoustic matching layer 4We have presented an approach to designing laser-induced ultrasound transmitters for large-volume ultrasound computed tomography. The transmitters produced according to our method behave in a satisfactory and reproducible manner, though some room for improvement in the process and result still remains. The LIUS transmitters produced here have a centre frequency of 0.94 MHz with a bandwidth from 0.17 to 2.05 MHz, producing pressures between 180.17 kPa and 24.35 kPa for a range of depths between 7.42 and 62.25 mm. The ultrasound field has a cylindrical symmetry about the acoustic axis and an opening angle of 45.8The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Ameliorating graft injury induced by ischemia and hypoxia, expanding the donor pool, and improving graft quality and recipient prognosis are still goals pursued by the transplant community. The preservation of organs during this process from donor to recipient is critical to the prognosis of both the graft and the recipient. At present, static cold storage, which is most widely used in clinical practice, not only reduces cell metabolism and oxygen demand through low temperature but also prevents cell edema and resists apoptosis through the application of traditional preservation solutions, but these do not improve hypoxia and increase oxygenation of the donor organ. In recent years, improving the ischemia and hypoxia of grafts during preservation and repairing the quality of marginal donor organs have been of great concern. Hemoglobin-based oxygen carriers (HBOCs) are “made of” natural hemoglobins that were originally developed as blood substitutes but have been extended to a variety of hypoxic clinical situations due to their ability to release oxygen. Compared with traditional preservation protocols, the addition of HBOCs to traditional preservation protocols provides more oxygen to organs to meet their energy metabolic needs, prolong preservation time, reduce ischemia–reperfusion injury to grafts, improve graft quality, and even increase the number of transplantable donors. The focus of the present study was to review the potential applications of HBOCs in solid organ preservation and provide new approaches to understanding the mechanism of the promising strategies for organ preservation. Solid organ transplantation (SOT) is an optimal, lifesaving treatment choice for patients with end-stage organ failure. The number of transplants has increased dramatically in the last decade, but there remains a considerable imbalance between the demand and supply of donor organs worldwide , 2021b. Ischemia and hypoxia are the leading causes of graft damage during the storage of isolated organs and may lead to a variety of destructive effects, such as ion imbalance, uncoupling of mitochondria, activation of endothelial cells, activation of various cell death programs, and proinflammatory immune responses . The curHBOCs are cell-free carriers obtained by processing free hemoglobin via chemical modification, genetic modification, or embedding . The oxy2O2-driven formation of ferryl myoglobin (Mb), which generates radicals and contributes to renal dysfunction  to have the ability to bind and release oxygen. Hb is a tetrameric protein molecule of ∼64 kDa consisting of two noncovalently bound αβ dimers . Free Hbfunction . Moreovefunction . Hb crosfunction . HBOCs wfunction . Howeverfunction . Subsequfunction . Since 2function . HBOCs afunction . Owing tfunction , initiatfunction , tumors function , and othArenicola marina (M101) (Arenicola marina), HBOC-201 (bovine), hemoglobin-vesicles (HbV) (human), polymerized human placenta hemoglobin (PolyPHb) (human), and hemoglobin glutamer (HbG) 200 (bovine). An overview of the different oxygen carriers used in solid organ preservation is provided to understand their properties better  to increase the stability and molecular size  , upregulAlthough there are differences in these vascular parameters, there are few reports on vasoconstriction damage in the preservation of isolated organs. The following reasons may underlie the relative lack of reports: carbon monoxide (CO) production is increased during the degradation of heme ; HBOCs pin vitro  , Celsiorin vitro . The appin vitro .Compared to other solid organs, the clinically isolated heart has a relatively short safe cold storage time (up to 4–6 h). The heart is an aerobic organ that relies on a continuous supply of oxygen to produce high-energy phosphate ATP for mechanical functions. The supply of oxygen via HBOCs supports the resynthesis of ATP in mitochondria of isolated heart tissue, which delays the damage of ischemia and hypoxia and extends the cold storage time . Li et aex vivo heart perfusion (EVHP) to meet the needs of myocardial metabolism, but it did not minimize myocardial injury or provide superior preservation of myocardial function  of the graft in the M101 group were significantly improved, and the graft vascular resistance was reduced after 5 h of reperfusion in the recipient. The study identified a circulating biomarker of ischemia–reperfusion, high mobility group box 1 protein (HMGB1). The expression of HMGB1 in the serum of the M101 group tended to be lower, and the activation of the HMGB1/RAGE (advanced glycation end products) axis in the control group explained the inflammatory response that led to PDG. The early serious events that occurred in the first few hours after transplantation were sufficient to capture the development of PDG. Therefore, the protective effect of M101 on IRI prevented the occurrence of PGD. Another study reported that SCS for up to 36 h after adding M101 reduced the risk of PGD after lung transplantation, which may be related to a significant decrease in circulating interleukin-6 (IL-6) levels (ex vivo lung perfusion (EVLP) for 12 h; the M101 lung showed better lung function after transplantation. M101 has inherent oxidative stress reduction activity, but it produces no significant difference in oxidative injury. The effect of M101 in prolonging the isolated lung’s preservation time may not be as an antioxidant, but it may optimize tissue oxygenation in the area of atelectasis. Briefly, adding M101 preservation solution prolongs the preservation time of isolated lungs up to 48 h, which will help overcome long-distance obstacles and expand the donor pool.Lung transplantation has been the most effective treatment for most end-stage lung diseases since 1983 . IRI is ) levels . CompareLiver graft quality depends on the donation status of the donor and the preservation effect during transportation from the donor to the recipient. During the preservation of the isolated liver, insufficient oxygen supply followed by reperfusion leads to an inflammatory network, and oxidative stress in the tissue leads to IRI. HBOCs induce the production of heme oxygenase 1 (HO-1) . Topp anNMP is generally performed using perfusion solutions based on RBCs to maintain physiological osmolarity, oncotic pressure, and adequate metabolic support. Laing et al. presented the first experience using an HBOC-based perfusion fluid to perfuse five discarded high-risk human livers in a human model of NMP . Livers 2) ± M101 (respectively or combined)] with HMP on the perfusion of isolated kidneys in a pig kidney auto-transplantation model  and maintained a high level of kidney injury molecule-1 (KIM-1) in the circulation. The study showed that supplementation with M101 with or without 100% O2 improves the HMP effect on kidney recovery and late graft results.Kidney transplantation is the therapy of choice for end-stage renal diseases. IRI is a complex process that involves oxidative stress, mitochondrial uncoupling, and the coagulation cascade, which affect early graft function after organ transplantation and have an adverse effect on the long-term survival of grafts . Thuillion model . The resGradual rewarming is a new perfusion modality in which HBOC maintains the preservation of perfusion of organs from hypothermia to normothermia. A transient increase in vascular resistance and methemoglobin was observed after preservation of the kidney using a preservation solution supplemented with HBOC-201 in a model of gradual rewarming but improved ultrafiltrate production, glomerular filtration rate, and sodium reabsorption during the reperfusion phase . In the Pancreas or islet transplantation is a treatment for type 1 diabetes and pancreatitis . During Muscle is a promising alternative site for islet transplantation, facilitating the rapid restoration of islet vasculature; however, it is susceptible to hypoxic limitation in the early posttransplant period. By co-transplanting islets with an oxygen carrier-PolyHb in mice, Daniel et al. found a dose-dependent reduction in β-cell hypoxia . Yet, thDue to the global shortage of organ resources, the imbalance of donor–recipient needs, and the increasing number of high-risk donors, these issues have led to considering what should be done to preserve grafts better. Although standard preservation solutions are currently used for static cold preservation, the repair and evaluation of marginal organs may rely on multiple combinations of MP, oxygenation strategies, and temperature regulation. Many efforts have been made by clinical practitioners and researchers to explore ways to mitigate graft ischemia–reperfusion injury, improve graft quality, and increase recipient and graft survival rates. Originally developed and utilized as a blood substitute, HBOCs are an ideal class of oxygen carriers with the ability to bind oxygen and release oxygen. HBOCs have now been extended to apply to tissues or organs in hypoxic conditions, where they act as an oxygen bridge to improve tissue oxygenation. The addition of HBOC to the preservation solution to improve the quality of the graft is a new promising strategy because they offer several advantages compared with traditional preservation solutions. First, HBOCs improve the oxygenation of isolated organs during SCS and enhance the quality of grafts. Second, the combination of HBOCs and MP provides a more targeted method, which can replace RBC perfusion to improve and evaluate the quality of high-risk grafts. In addition, preservation solutions containing HBOCs may be easily used in all solid organ transplants without further improving the existing preservation solution or perfusion platform protocols. Recent clinical trials have demonstrated the safety and efficacy of HBOCs in combination with mechanical perfusion platforms for liver and kidney preservation and improved prognosis for recipients and grafts.Future prospects address the need for translational medicine research to determine the exact therapeutic dose and organ preservation time for human solid organs. Coordinated multicenter clinical trials are also needed in the subsequent clinical studies to accumulate larger, homogeneous cohorts. Additionally, some clinical trials with a randomized controlled design would be performed. This review summarizes the latest and most important research on HBOCs in solid organ preservation. Therefore, based on scientific evidence, HBOCs, one of the oxygen carriers for organ preservation, are ideal candidates for organ preservation additives. The mechanism of organ preservation by HBOCs has not been fully understood; the relevant mechanism would be investigated in depth in future studies to determine a more optimal preservation method. In addition, the vascular resistance, methemoglobin content, and oxidative damage index of grafts and recipients would also be closely monitored when using HBOCs. The future development of HBOCs may give a fascinating insight into the understanding of the safety and effectiveness of new strategies for solid organ preservation.


Reason: The article, Mlenga, D.H., Jordaan, A.J. & Mandebvu, B., 2019, ‘Monitoring droughts in Eswatini: A spatiotemporal variability analysis using the Standard Precipitation Index’, Jàmbá: Journal of Disaster Risk Studies 11(1), a725. https://doi.org/10.4102/jamba.v11i1.725, published on 03 October 2019 in Jàmbá - Journal of Disaster Risk Studies has been retracted by agreement between the authors , the Publisher (AOSIS) and the journal’s Editor-in-Chief, Prof. Dewald van Niekerk, because of duplicate publication. The second article was published on 24 October 2019 in the Jàmbá - Journal of Disaster Risk Studies (https://doi.org/10.4102/jamba.v11i1.712).Jàmbá - Journal of Disaster Risk Studies.We apologise for any inconvenience to the authors and readers of


In severe prostatitis, csPCa was determined in only 7.7% (4/52) of PI-RADS 3 lesions. In severe chronic prostatitis, a binary prostatitis suffix  within the radiological report may help assess the limitations of mpMRI interpretability because of severe prostatitis and avoid unnecessary biopsies. Mean apparent diffusion coefficient (ADCmean) was the best marker (cutoff 0.93 × 10−3 mm2/s) to differentiate between csPCa/non csPCa in severe prostatitis.We examined the impact of chronic prostatitis on the diagnostic performance of multiparametric magnetic resonance imaging (mpMRI). In this retrospective study, 63 men underwent 3T mpMRI followed by MRI/ultrasound fusion biopsy to exclude/confirm clinically significant prostate cancer (csPCa). A total of 93 lesions were included for evaluation. Images were assessed by two radiologists. Prostatitis was graded visually on T2-weighted and contrast-enhanced sequences. The correlation of prostatitis features with the assigned Prostate Imaging Reporting and Data System (PI-RADS) and the presence of csPCa were assessed, and the clinical and functional imaging parameters for differentiating between prostatitis and significant tumors were examined. Histopathological analysis was used as the reference standard. The rate of PI-RADS 3 scores tended to be higher in the presence of radiologically severe prostatitis compared with no/discrete prostatitis ( Multiparametric magnetic resonance imaging (mpMRI) is an important clinical tool for the detection and guidance of treatment for prostate cancer (PCa) ,2,3,4. AChronic prostatitis is a very common disease in older people and usually progresses without symptoms. Fluctuating and slightly elevated prostate-specific antigen (PSA) levels are often detected by the urologist or general practitioner, which necessitates further work-up with MRI to rule out PCa ,9. In imIn PI-RADS v2, the presence of prostatitis may reduce diagnostic accuracy . Neverth2, respectively. The predefined exclusion criteria are detailed in Between April 2019 and December 2019, 63 patients (64.3 ± 5.7 years) who received a clinically indicated mpMRI of the prostate followed by an MRI/ultrasound (US) fusion biopsy to rule out or confirm PCa were included in this retrospective study. The mean PSA, prostate volume, and mean PSA density were 9.69 (1.03–139.00) µg/L, 66.2 (20.6–182.0) mL, and 0.165 (0.015–2.000) ng/mL2; slice thickness, 3 mm; FOV, 170 mm; TR, 4900 ms; TE, 58 ms) with a calculated ultrahigh b-value (b = 2000 s/mm2) and a parametric ADC map, and a native T1 and prototypical dynamic T1 gradient echo sequence (csVIBE) for DCE with high temporal resolution . The csVIBE was acquired during free breathing after administration of 0.1 mmol/kg gadopentetate dimeglumine  at a rate of 2 mL/s and subsequently reconstructed by compressed sensing. All examinations were performed with a clinical 3T MRI scanner  equipped with a 50-channel coil setup (18-channel body coil and 32-channel spine coil). Patients were positioned head-first supine in the MRI machine. The MRI protocol was designed according to the recommendations of the PI-RADS v2.1 guidelines and consisted of high-resolution T2W Turbo Spin Echo sequences , 180 mm; repetition time (TR), 4000 ms; echo time (TE), 112 ms), DWI using the RESOLVE sequence  and 5 years (S.M.) of experience in prostate MRI graded the prostatitis independently using the axial T2W and the DCE according to the scheme shown in From this assessment, we derived an algorithm for a simple inflammation suffix, as follows: mild findings in T2W were labeled as ‘i−’ (no inflammation) but were upgraded to ‘i+’ (inflammation) if DCE showed severe enhancement, while severe findings in T2W were labeled as ‘i+’ regardless of DCE . We assu2 circle shape) and was only altered if tissue obviously located outside of the lesion would have been included. The minimum, maximum, and mean ADC values were measured in each lesion with a PI-RADS score ≥3. The measured area was kept constant (11.5 mmtrans (volume transfer rate reflecting the efflux rate of gadolinium contrast from blood plasma into the tissue extravascular extracellular space (EES), Kep (time constant for gadolinium contrast reflux from EES back into blood plasma), Ve , area under the curve (AUC), and Chi-square values [The csVIBE sequence was used to analyze the contrast kinetics. We applied the two-compartment Tofts model, where a systemically administered contrast agent penetrates a capillary wall of increased permeability into the interstitium. The amount of contrast accumulation in the interstitium is dependent on capillary permeability, the difference in the concentration on either side of the capillary wall, and on the total surface area . In neope values , as welle values .The same experienced radiologist (12 years of experience) reported all prostate MRI examinations and subsequently performed the lesion segmentation . MRI conventional transrectal ultrasound-guided biopsies were performed by urology consultants with a median experience of 5 years using an Artemis device  according to the European Association of Urology guidelines .Depending on the lesion sizes, between 2 and 4 cores of each target lesion and complementary template biopsies of the remainder of the prostate  were sampled. This lesion-based histopathological evaluation served as the gold standard for our examination. Clinically significant cancer was defined as a Gleason score ≥4 + 3 (Grade 7b and higher) or a maximum cancer core length of ≥6 mm according to the Epstein criteria . Only bit-tests were used to determine variables with discriminating power for the different clinical and functional imaging parameters , log(PSAlatest)) and were judged by their respective p-values. Bonferroni correction was applied to account for multiple testing ). A p-value of <0.05 was considered statistically significant.Only the ratings of the experienced radiologist were used for the statistical evaluation. The ratings of the less experienced radiologist were used to determine the interobserver agreement using Cohen’s kappa. The Chi-square test was used to evaluate the correlation between PI-RADS scores and prostatitis. Here, the PI-RADS 4 and 5 lesions were combined, and no corrections were used. An approximately normal distribution was verified in pair plots with bell-shaped histograms. Where this was not the case, the logarithm was used . Univariate two-sample mean for the differentiation between csPCa and non-csPCa/no tumor was used for no/discrete prostatitis and severe prostatitis. Sensitivity, specificity, and corresponding cutoffs were calculated. All statistical analyses were performed with statistical software (Python (www.python.org), using sklearn, numpy, and matplotlib libraries for ROC analysis) .Using logistic regression, the area under the receiver operating characteristic (ROC) curve for univariate observation of ADCAmong the 93 lesions in the PZ, csPCa was histopathologically confirmed in the targeted biopsy in 18 lesions. Insignificant cancer was found in 12 target lesions. The 63 remaining targeted biopsies were negative. On the template biopsy, only one additional csPCa was detected, although no significant tumor was found in the target biopsy.n = 31; severe with enhancement, n = 22) and as not evident/discrete in 11 patients. Cohen’s kappa for inter-reader agreement was substantial for both groups . Overall, 65.6% of lesions (61/93) were classified as PI-RADS 3, 25.8% (24/93) as PI-RADS 4, and 8.6% (8/93) as PI-RADS 5. Chronic prostatitis was rated as severe in 53 patients , especially if there was also an increased contrast enhancement as an indicator of inflammatory activity  compared with no/discrete prostatitis  were the best discriminators between csPCa and non-significant PCa/no tumor , 0.76–1.00) in no/discrete prostatitis and 0.92  in severe prostatitis. The AUCs were not improved using multivariate ROC curves  in no/discrete prostatitis and 0.91  in severe prostatitis)  . mean), the sensitivity for csPCa was 1 for either no/mild prostatitis and severe prostatitis, with a specificity of 0.83 and 0.76, respectively. The ADCmean cutoff for differentiating between csPCa/non-csPCa was 0.9282 × 10−3 mm2/s for no/mild prostatitis and 0.9285 × 10−3 mm2/s for severe prostatitis. In the multivariate assessment , the sensitivity for csPCa was also 1 for either no/mild prostatitis and severe prostatitis, with a specificity of 0.83 and 0.79, respectively. Note that because of the small sample size , there was insufficient power for this evaluation.In the univariate assessment , while csPCa was rarely detected histopathologically in these PI-RADS 3 lesions (7.7% (4/52); −3 mm2/s, 1.08 ± 0.18 × 10−3 mm2/s, 0.88 ± 0.15 × 10−3 mm2/s, and 0.88 ± 0.13 × 10−3 mm2/s, respectively. In that study, there were significant differences between prostatitis and both high- and low-grade PCa (p < 0.001), although there was a clear overlap of ADC values [−3 mm2/s achieved the highest predictive value for prostatitis  [−3 mm2/s for differentiating between csPCa and non-csPCa.In the current version of the PI-RADS classification system (PI-RADS v2.1) , DWI is C values . In anot, 0.859) . This isSeveral studies have examined whether prostatitis and PCa can be discriminated with PI-RADS. A study by Zhang et al. of 77 patients with PCa and 29 with prostatitis showed that prostatitis and csPCa of the PZ could be distinguished by PI-RADS v1 and v2 . Liddelltrans, kep, Ve, and Vp), the lesion-to-normal prostate tissue ratios, and time-to-peak (TTP) values could successfully differentiate between PCa and prostatitis [n = 32) [Interestingly, Rourke et al. showed that inflammation can mimic PCa, with almost 45.9% of biopsies being negative for malignancy, although the PI-RADS score for each lesion was not assigned . In contstatitis . Further[n = 32) .ep, adj. p = 0.32; Ktrans, adj. p = 1; It remains controversial whether quantitative DCE-MRI parameters improve the diagnostic performance of mpMRI for distinguishing between normal tissue and PCa, including differentiation of cancer grades . Furthervelocity, adj. p = 1; log(PSAdensity), adj. p = 0.38; log(PSAlatest), adj. p = 1) did not show a significant benefit for discrimination between csPCa and non-csPCa. Polanec et al. investigated the potential to integrate clinical and biochemical patient parameters to avoid unnecessary MRI-guided biopsies. In that study, patient characteristics , biochemical parameters , and PI-RADS v2 scores were evaluated, with the combined regression model  providing an AUC of 0.84 to predict csPCa . Furthermore, biopsies could have been avoided in 50% of patients (64/128), while csPCa would have been missed in only 4% (2/50) [We also found that the biochemical parameters (PSA% (2/50) . In the Our study has some major limitations. First, the prostatitis grading was performed visually based on the observation of T2W and DCE, while histopathological evidence was not obtained. Long-term PSA levels were not evaluated. Furthermore, our cohort was too small to detect statistically significant differences between the groups for the association between prostatitis, csPCa/non-csPCa, and PI-RADS 3 scores. In this context, the groups with PI-RADS scores of 4 and 5 and only low/no prostatitis were very small.We found that PI-RADS 3 lesions of the PZ often correspond to prostatitis and that after MRI/US-guided biopsy, these areas rarely show a csPCa. We believe that this is a diagnostic issue leading to significant limitations in the interpretability of mpMRI, which has not been sufficiently appreciated in PI-RADS to date. To address this problem, we recommend that a prostatitis suffix be implemented in the radiological report for PI-RADS 3 findings in the PZ. We suggest a binary nomenclature for inflammation with the suffix i+ and i− (e.g., PI-RADS 3 with significant inflammation = PI-RADS 3 i+; PI-RADS 3 without significant inflammation = PI-RADS 3 i−; Furthermore, in the PZ, ADC was the best marker in our small cohort to reliably differentiate between csPCa and benign changes or non-significant tumors, regardless of the degree of prostatitis. This underlines the importance of establishing an ADC threshold to avoid unnecessary PI-RADS 3 scores and biopsies and to improve interobserver agreement and diagnostic confidence.


The consumption of good tasting rice, mainly soft-textured white rice with low amylose content, has substantially increased in China as living standards improve. However, this diet change may increase the risk of developing type II diabetes because the soft-textured white rice is generally less resistant to digestion and has a higher glycemic index. In contrast, intake of brown rice is inversely associated with type II diabetes risk. This study was conducted to test the possibility that brown rice processed from soft-textured cultivars has both acceptable texture and improved health benefits. Texture and digestion properties were compared between white and brown rice of five indica cultivars preferred by Chinese consumers. Mean hardness was 33% higher while mean springiness was 5% lower for cooked brown rice than for cooked white rice. As compared to cooked white rice, cooked brown rice had a 41% longer mean active digestion duration but 31% lower mean glucose production rate and 11% lower mean total glucose production from starch digestion. However, the differences in texture and starch digestion properties between cooked brown and white rice were affected by cultivar identity. Brown rice processed from suitable cultivars with both a relatively thinner bran layer and relatively higher grain amylose content met consumer requirements in terms of acceptable texture and improved health benefits. China has been self-sufficient in rice supply due to an increase in grain yield per unit area by more than 50% since 19802. The sufficient supply of rice has led to dietary changes such as replacing coarse cereals  and whole grains  with polished rice 4. More recently, as living standards improve in China, the production and consumption of good tasting rice, mainly soft-textured white rice with low amylose content, has substantially increased6.China has the largest population of rice consumers in the world, with more than 65% of the population (~ 900 million people) eating rice as a staple food8. The increased consumption of low amylose, soft-textured white rice may increase the risk of developing type II diabetes because this type of rice is generally less resistant to digestion and has a higher glycemic index9. On the contrary, intake of coarse cereals and whole grains including brown rice is inversely associated with type II diabetes risk11. In addition, whole grain foods have other health benefits, such as reducing the risk of cardiovascular disease and cancer13, which are of increasing concern both nationally and globally due to their high mortality rates14.There is ample evidence that consuming more white rice is associated with an increased risk of type II diabetes in Asian populations including the ChineseBy comprehensively considering consumer preferences for soft-textured rice and the health benefits of brown rice, we speculated that brown rice processed from soft-textured cultivars may have both acceptable texture and improved health benefits. To test this, we conducted a study comparing the texture and digestion properties of white and brown rice of five indica cultivars preferred by consumers in China.Averaged across five cultivars, the milling recovery rate was 21% higher for brown than for white rice  of cooked rice of tested cultivars. The decreased eating quality of cooked brown rice compared to cooked white rice was mainly attributable to the retained fiber-rich bran layer18. However, in this study, the harder texture of cooked brown rice compared to white rice was not explained by the difference in grain amylose content, because brown rice had lower grain amylose content compared to white rice.Grain amylose content is also a key determinant of the eating quality of cooked rice, and higher grain amylose content usually leads to a harder texture of cooked rice11, since rice starch with higher resistance to digestion has a lower glycemic index21. Grain amylose content is a critical component determining the starch digestion rate of cooked rice, and higher grain amylose content is always associated with a higher resistant starch content and a lower starch digestion rate9. However, this was not responsible for the slower starch digestion rate in cooked brown compared to white rice in this study, because the grain amylose content was lower in brown than in white rice.The starch digestion of cooked rice was generally slower due to shifting from white to brown rice. This could explain why intake of brown rice is inversely associated with the risk of developing type II diabetes15. These non-starch components may complex with starch and inhibit starch digestion by affecting starch properties; producing a protective layer around starch; or having antagonistic effects on digestive enzymes22. For example, protein present in endosperm can decrease the starch digestion rate of cooked rice by restricting swelling and reducing the surface area of the starch granules24. Therefore, in this study, the slower starch digestion of cooked rice due to shifting from white rice to brown rice was mainly attributable to the retained bran layer that is rich in non-starch components such as protein.The starch digestion rate of cooked rice also depends on non-starch components such as fiber, protein, lipids, and polyphenols, which are rich in the bran layer of brown riceIn addition, this study showed that the differences in texture and starch digestion properties between cooked brown and white rice were affected by cultivar identity. In particular, cooked brown rice of Meixiangzhan 2, Nongxiang 42, and Taoyouxiangzhan had lower hardness and chewiness than cooked white rice of Yuzhenxiang and a slower starch digestion rate than cooked white rice of all cultivars. This outcome indicates that selecting suitable cultivars such as Meixiangzhan 2, Nongxiang 42, and Taoyouxiangzhan and processing their grains into brown rice is expected to provide acceptable texture and benefit health. Based on grain quality traits, Meixiangzhan 2, Nongxiang 42, and Taoyouxiangzhan had a relatively thinner bran layer  and relatively higher grain amylose content among the five tested cultivars Table . This fi26. Moreover, there are other grain quality traits, such as aroma and appearance, that determine consumer preference for rice27. Therefore, more studies including consumer organoleptic tests and clinical trials are required to comprehensively evaluate the acceptability and feasibility of brown rice processed from soft-textured cultivars.There are some limitations that should be acknowledged. This study evaluated the texture acceptability and health benefits of brown rice only by comparing its physicochemical properties with those of white rice, but not by direct consumer involvement. In this regard, it is advocated that the consumer involvement can play a vital role in food and health researchShifting from white to brown rice retains the bran layer with abundant non-starch components such as protein, and consequently leads to decreased eating quality  but an increase in health quality  of indica cultivars preferred by Chinese consumers. However, brown rice with acceptable texture and improved health benefits can be obtained by selecting suitable cultivars  that have a relatively thinner bran layer and relatively higher grain amylose content.Rice grains of five indica cultivars—Meixiangzhan 2, Nongxiang 42, Taoyouxiangzhan, Xiangyaxiangzhen, and Yuzhenxiang—were collected from the research base of the Hengyang Academy of Agricultural Sciences , Meihua Village, Hunan Province, China in 2020. These rice cultivars have good taste and a soft texture. In particular, four of the five cultivars  won the Gold Award of the Eating Quality Evaluation Contest of High-Quality Indica Rice in China. The use of plants in the present study complies with international, national and/or institutional guidelines.28. Milling recovery rates of brown and white rice were calculated by separately dividing the brown and white rice weight by the grain weight.Rice grain samples were air-dried and then stored for three months before analysis. For each cultivar, 100 g of rice grain samples were de-hulled to obtain brown rice and then half of the brown rice was polished to white rice, using a laboratory-scale milling machine  and closely following the procedure of the International Standard ISO 6646:201129. In brief, amylose content was measured with the iodine colorimetric method. The protein content was determined by multiplying N content by a conversion factor of 5.95; N content was measured with an automatic Kjeldahl analyzer .About 5 g of brown and white rice flours (filtered through 100 mesh) were prepared for each sample to determine amylose content and protein content according to procedures described by Huang et al.+; Shanghai Tengba Instrument Technology Co. Ltd., Shanghai, China).Approximately 10 g of white and brown rice were soaked in 16 ml of distilled water in an aluminum cup for 30 min, and the aluminum cup was then covered with a lid and placed in an electric rice cooker  containing approximately 700 ml of boiling water and allowed to steam for 40 min. The lid of the electric rice cooker remained locked for 20 min after steaming was complete. Texture profiles  of the cooked rice were determined using a texture analyzer  using a Glycemic Index Analyser . The starch digestion process of the cooked rice  was fitted to an exponential association model, y = a[1 − EXP(− bx)], based on goodness of fit and biological plausibility . The digestion parameters of the cooked rice, including active digestion duration, total glucose production, and glucose production rate, were estimated with y at 95% of a (0.95a) using the following formulas: active digestion duration = LN(0.05)/− b; total glucose production = 0.95a; and glucose production rate = total glucose production/active digestion duration; a and b were obtained from the fitting.t-tests . Statistical significance was set at the 0.05 probability level.All data were compared between brown and white rice by paired


Brassica rapa. The stigma- and pollen-side recognition factors  of this UI are similar to those of SI (stigma-side SRK and pollen-side SP11), indicating that SUI1 and PUI1 interact with each other and cause pollen-pistil incompatibility only when a specific genotype is pollinated. To clarify the genetic diversity of SUI1 and PUI1 in Japanese B. rapa, here we investigated the UI phenotype and the SUI1/PUI1 sequences in Japanese commercial varieties of Chinese cabbage. The present study showed that multiple copies of nonfunctional PUI1 were located within and in the vicinity of the UI locus region, and that the functional SUI1 was highly conserved in Chinese cabbage. In addition, we found a novel nonfunctional SUI1 allele with a dominant negative effect on the functional SUI1 allele in the heterozygote.In recent years, unilateral incompatibility (UI), which is an incompatibility system for recognizing and rejecting foreign pollen that operates in one direction, has been shown to be closely related to self-incompatibility (SI) in  Brassica rapa L.) are F1 hybrids. Traditionally, their seeds have been produced using the Brassica self-incompatibility (SI) system. The SI system in Brassica is sporophytically controlled by a single S-locus with highly variable, multiple alleles [S-locus region contains two genes, SRK and SP11/SCR, which correspond to female and male S determinants, respectively [SRK encodes a transmembrane receptor kinase, which is expressed specifically in stigma, and SP11/SCR encodes a small cysteine-rich ligand for SRK, which is localized on the pollen coat [S-haplotype-specific interaction of SP11 and the extracellular domain of SRK induces the SI reaction, in which the self-pollen fails to germinate or penetrate into the stigma [S-haplotypes has been estimated to be more than 100 in B. rapa [S-haplotype diversity, including dominance relationships between the haplotypes [1-hybrid seed in Brassica crops.Most Japanese cultivars of Chinese cabbage  is on chromosome A04 of B. rapa [PUI1 and SUI1 genes in B. rapa, a functional PUI1-1 allele was found only in the Turkish lines and not in the Japanese lines, while the three functional SUI1 alleles  were found in Japanese wild populations and some cultivated varieties. Thus, loss of function of SUI1 in Turkish lines and PUI1 in Japanese lines might have resulted in the unidirectional pollen-stigma incompatibility in B. rapa [In addition to SI, we reported an interesting incompatibility relationship between Turkish and Japanese populations of B. rapa ,12. PollB. rapa ,14. Our M-locus protein kinase (MLPK) in UI, which may function in SRK-mediated SI signal transduction [B. rapa. Thus, UI may have an effect on the SI-dependent breeding process in B. rapa. In this study, we extensively analyzed SUI1 and PUI1 alleles in Japanese cultivated lines of Chinese cabbage (Brassica rapa var. pekinensis). The results presented here give new insight into the historical relationship between UI and the breeding system of Chinese cabbage in Japan.The physiological pollen-rejection phenotype of the intraspecies UI is similar to that of SI and is consistent with the involvement of sduction ,16. It iB. rapa var. perviridis), from the Takii seed company [SUI1 in Japanese B. rapa cultivars, here we examined 52 commercial cultivars of Chinese cabbage (B. rapa var. pekinensis) from 16 Japanese seed companies , are F1 hybrids. Because functional SUI1 alleles behave as dominant over nonfunctional alleles [The UI phenotype observed on the stigma (stigma-side UI phenotype) was originally identified in the Japanese commercial hybrid variety ‘Osome’ of Japanese mustard spinach, Komatsuna  possessing PUI1-1/PUI1-1 with crossing combinations of different S-haplotypes for discriminating the UI phenotype from the SI phenotype (21tS was produced for this study) [PUI1-1/PUI1-1 pollen, indicating that they possess functional SUI1 allele(s).To verify the stigma-side UI phenotype of the Japanese cultivars of Chinese cabbage, stigmas of 47 cultivars were crossed with the pollen from the Turkish line (s study) . Among tSUI1 gene by polymerase chain reaction (PCR) amplification from the genomic DNA of each cultivar and determined its allele(s) by sequencing, as listed in SUI1 was sequenced, six alleles, including functional alleles (SUI1-1 and -2), were identified. One cultivar, ‘Kashinhakusai’ (#8), with stigmatic UI phenotype, had the SUI1-1 allele, which was originally isolated from Komatsuna variety ‘Osome’ [1 hybrid, as described above. The 16 cultivars with stigmatic UI phenotype possessed the SUI1-2 allele , SUI1-11 , and SUI1-12 , and one allele encoding truncated protein, sui1-t10  were newly identified alleles in this study  and SUI1-10  and ‘Taibyou apolo 60′ (#83), a single base substitution at codon 413 (changing the residue from cysteine to tyrosine) was present at the C-terminus of the extracellular domain .In the case of the r domain . Both cu2 allele , indicatSUI1-11 allele had 17 amino acid changes in the extracellular domain, and cultivars ‘Mainoumi’ (#1) and ‘Menkoi’ (#9) with SUI1-2/SUI1-11 heterozygote showed the stigmatic UI phenotype  had SUI1-2 and SUI1-12 alleles as a heterozygote, and it showed the stigmatic UC phenotype (SUI1-12 might show a dominant negative effect to SUI1-2 in ‘Kasumihakusai’ (#17), as in the case of SUI1-10 in ‘Hakuei hakusai’ (#50) and ‘Taibyou apolo 60′ (#83).The henotype , Table 1sui1-t10 allele (SUI1 clones of ‘Chihiri 70′ (#5) isolated from two independent PCR amplifications were sui1-t10, suggesting that ‘Chihiri 70′ (#5) is homozygous for sui1-t10, which is consistent with its stigmatic UC phenotype. The sequence of the extracellular domain of sui1-t10 was perfectly matched with SUI1-1 and SUI1-2 functional alleles, but there was a 10-bp deletion in the sixth exon, as in sui1-t4, sui1-t5, and sui1-t6, which results in a frameshift and creates a premature termination codon [‘Chihiri 70′ (#5) possessed the truncated 0 allele . All theon codon .PUI1 alleles of 48 cultivars of Chinese cabbage, we cloned the PCR fragments of the full-length PUI1 and determined their sequences , which have been reported previously [pui1-3 and pui1-4, and 14 possessed all three alleles  and determined the PUI1 genotype of the 22 segregants using a direct sequencing method. It was found that all the segregants exhibited both pui1-3 and pui1-4, suggesting that the pui1-3 and pui1-4 genes were linked and homozygous in this progeny (pui1-3/pui1-4/pui1-6) and determined the PUI1 genotype of the 32 segregants using a PCR-restriction fragment length polymorphism (RFLP) method. It was found that all individuals possessed pui1-3, pui1-4, and pui1-6, suggesting that the three PUI1 genes (pui1-3/pui1-4/pui1-6) were linked and homozygous in this progeny (pui1-3/pui1-4/pui1-6) selfed progeny  fit for a simple Mendelian inheritance. The result indicates that ‘Nanzan’ (#101) is a heterozygote of duplicated (pui1-3/pui1-4) and triplicated (pui1-3/pui1-4/pui1-6) PUI1 genes in the B. rapa genome. Thus, duplication and/or triplication of nonfunctional PUI1 genes had occurred at the UI locus region in Japanese B. rapa cultivars.To examine the eviously . Out of  alleles . The exi progeny . Next, w progeny . Further progeny . InteresSUI1-2/SUI1-10 genotype, exhibited stigma-side UC phenotype , even though they have a functional SUI1-2 allele. To confirm this dominant negative effect of SUI1-10, we performed a genetic analysis of ‘Taibyou apolo 60′ (#83).As described above, ‘Hakuei hakusai’ (#50) and ‘Taibyou apolo 60′ (#83), possessing the 1 progeny) and determined their stigma-side UI phenotype and SUI1 genotype (PUI1-1/PUI1-1 homozygous plants (S24t) as the pollen donor. The SUI1-2 and SUI1-10 alleles were discriminated by direct-sequencing detection of a single nucleotide polymorphism at codon 413 and were segregated in the #83-S1 progeny; three of eleven plants showed stigma-side UI, and the others were stigma-side UC. Stigma of thee SUI1-2/SUI1-2 homozygous plants were incompatible to the S24t pollen (UI), and five SUI1-2/SUI1-10 heterozygous, and three SUI1-10/SUI1-10 homozygous individuals showed compatible pollen tube penetration with the S24t pollen (UC), indicating that SUI1-10 is nonfunctional and has a dominant negative effect to the functional SUI1-2.We produced self-pollinated progeny of ‘Taibyou apolo 60′  with PUI1-1/PUI1-1 pollen from the S24t and also S40t lines, indicating that SUI1-10 also shows a dominant negative effect on the functional SUI1-3.To verify if this effect is observed with the other functional allele, we produced 3/SUI1-3 ,12 and S1 hybrid seed production of Brassica cultivars. The molecular mechanism of SI in Brassica has been studied for many years and is used in F1 breeding. The recently discovered UI system, regulated by SUI1 and PUI1, can potentially provide another mechanism to control pollination in B. rapa. Therefore, determination of the UI genotype is considered as important as the SI genotype in the breeding of this major Japanese vegetable, Chinese cabbage. In this study, we determined the SUI1 and PUI1 allelic diversity of 22 and 48 cultivars, respectively, of Chinese cabbage in Japan. In addition, we confirmed the stigma-side UI phenotype of 47 cultivars. This revealed that most of the cultivars showed a stigma-side UI phenotype with a functional SUI1 allele (SUI1-2), whereas no functional PUI1 allele (PUI1-1) was found. We also searched the re-sequence data of B. rapa lines that are stocked at Chungnam National University and found a functional SUI1-2 allele in a South Korean population (data not shown). The fact that functional SUI1 alleles are present in Japanese and South Korean cultivars should be taken into consideration in breeding programs for B. rapa. UI may be beneficial as the additional incompatibility, which could be used in breeding programs by the introduction of PUI1-1 to the pollen donor.Highly controlled pollen-stigma incompatibility is important for FUI locus in chromosome A04. Thus, for an unknown reason, the functional SUI1-2 has been selected, and its sequence has been conserved during the breeding of Chinese cabbage cultivars in Japan. It would be interesting to investigate whether SUI1 itself strengthens SI and thus increases the efficiency of F1 seed production.To the best of our knowledge, there is no report that traits important for Chinese cabbage are mapped to flanking regions of the SUI1 and showed that SUI1-1, SUI1-2, and SUI1-3 are incompatible with PUI1-1/PUI1-1 pollen [SUI1-1 was originally isolated from a Japanese commercial hybrid variety of Komatsuna (B. rapa var. perviridis), and SUI1-2 and SUI1-3 were found in Japanese wild populations of B. rapa [SUI1 alleles; one (SUI1-10) belongs to the functional clade  and the other two alleles (SUI1-11 and SUI1-12) belong to the nonfunctional clade  compared to the extracellular domain of SUI1-2, suggesting that these four residues are not important for the effect.A feature of the sporophytic regulation of SI is the dominance relationship between plotypes ,22,23. Tgulation ,25,26. Ogulation . It was pression  noted thpression . On the pression ,28,29. IPUI1 gene of Japanese cultivars of Chinese cabbage showed very low diversity. Among six PUI1 alleles, of which only PUI1-1 from a Turkish strain can induce UI [pui1-3/pui1-4 or pui1-3/pui1-4/pui1-6) were observed, and no cultivars with a functional PUI1-1 allele could be found. Interestingly, the pui1-3/pui1-4 genotype might consist of two linked pui1-3 and pui1-4 genes , de novo genomic sequence assembly of these Chinese cabbage cultivars using next-generation sequencing technology, including long-read sequencing, would provide new insights into the genomic structure of the UI locus [PUI1 genes on the UI locus of the genome sequence of B. rapa Z1, such as turnips (subsp. rapa), leafy Brassica crops , and field mustard (subsp. oleifera) will not only contribute to the discovery of novel alleles but also provide new insights into the genomic structure of the pollen-side factor and the dominant recessive interaction of the stigma-side factor. It will also be interesting to determine whether the UI locus has a multi-allelic structure like the S locus.Further analysis of the genetic diversity of the B. rapa ssp. pekinensis , and the immature pistil was pollinated. The pollinated pistil was then covered with a paper bag until the seed was harvested. Plant materials were vernalized at 4 oC for 4 weeks in a refrigerator and then grown in a greenhouse.The plant material consisted of 52 commercial cultivars of Chinese cabbage, kinensis . All exc3PO4, 0.1% aniline blue). Samples were mounted in 50% glycerol on slides and observed by UV fluorescence microscopy  were used as the pollen donor in test pollinations (21tS was produced for this study) [Flower buds were cut at the peduncle and pollinated. After pollination, they were stood on 1% solid agar for about 24 h under room conditions. Then, pistils of the pollinated flowers were softened in 1N NaOH for 1 h at 60 °C and stained with basic aniline blue  .B. rapa by the procedure of Murray and Thompson (1980) or using a DNeasy plant mini kit (Qiagen) [SUI1 and PUI1 genes, genomic PCR was performed using KOD-Plus-Neo DNA polymerase (TOYOBO) according to the manufacturer’s instructions. PCR primers SUI1cDNA_F3 and SUI1_gR2 for SUI1 and PCP-like1-F1 and PCP-like1-R1 for PUI1 were used , followed by checking on an electrophoresed agarose gel  . For molere used . All ampnter_cF5 , were us0typeSDR . Each PU-3.4.6-R . For disrose gel . For thehttp://www.phylogeny.fr/, accessed on 21 October 2021) [Phylogenetic analysis was performed on the Phylogeny.fr platform (er 2021) . Full-le


Increases in the inflammatory mediators PTX3 (p = 0.048), CXCL8/IL-8 (p = 0.017), TNF-α (p = 0.038), and ANGPTL4 (p = 0.010) were observed, whereas the anti-inflammatory cytokine IL-10 was reduced (p = 0.007). Peculiar inflammation, cell death, and coagulation signatures were observed in fluids collected from DCD livers compared to those from DBD grafts. AST (p = 0.034), ALT (p = 0.047), and LDH (p = 0.047) were higher in the “EAD-yes” compared to the “EAD-no” group. Cytolysis markers and hyaluronan correlated with recipient creatinine, AST, and ICU stay. The study demonstrates that effluent molecular analysis can provide directions about the use of HOPE.Hypothermic-oxygenated-machine-perfusion (HOPE) allows assessment/reconditioning of livers procured from high-risk donors before transplantation. Graft referral to HOPE mostly depends on surgeons’ subjective judgment, as objective criteria are still insufficient. We investigated whether analysis of effluent fluids collected upon organ flush during static-cold-storage can improve selection criteria for HOPE utilization. Effluents were analyzed to determine cytolysis enzymes, metabolites, inflammation-related mediators, and damage-associated-molecular-patterns. Molecular profiles were assessed by unsupervised cluster analysis. Differences between “machine perfusion (MP)-yes” vs. “MP-no”; “brain-death (DBD) vs. donation-after-circulatory-death (DCD)”; “early-allograft-dysfunction (EAD)-yes” vs. “EAD-no” groups, as well as correlation between effluent variables and transplantation outcome, were investigated. Livers assigned to HOPE ( The adoption of expanded criteria for acceptance of organ donors broadened the pool of livers available for transplantation. However, relative to standard livers, organs procured from either brain death (DBD) extended criteria (ECD) or donation after circulatory death (DCD) donors are associated with enhanced risk of postoperative complications and transplant failure ,2,3. In In clinical practice, the decision to accept a marginal graft and to perform ex situ perfusion is based on both donor and graft characteristics, including occurrence of steatosis, cold ischemia time (CIT), warm ischemia time (WIT), donor brain or cardiac death, donor age, and donor comorbidities . AlthougThe analysis of effluent fluid, which is released upon organ flush on the bench and includes the residual preservation solution, could provide useful information to implement selection criteria for the livers that might benefit from further evaluation during MP. In fact, both clinical ,29,30,31We investigated if the assessment of specific factors in effluent fluids can help clinicians with decision making regarding the allocation of marginal graft to MP. In particular, we elected to assess the effluent concentration of biomarkers of hepatocellular damage, inflammation-related mediators, and DAMPs, which have been previously associated with brain death-induced inflammation, liver IRI, and poor transplantation outcome ,39,40. Tn = 49) who received a liver graft procured from either DBD or DCD donors from February 2017 to May 2019 in our center. Late re-LT and patients with incomplete data were excluded (n = 11).The present research includes all consecutive adult patients , the cava vein was clamped and grafts were flushed through the portal vein with 1000 mL of Celsior Solution . Effluent samples were collected through a 16G ago-cannula inserted in the posterior portion of the retrohepatic cava vein .Grafts were categorized as ECD based on the criteria described by Vodkin and coworkers . Donor aThe grafts were allocated to our center according to the North Italian Transplant program allocation policy. Our surgical team procured all grafts, with the exception of 4 national or interregional allocated livers . AlternaAll grafts underwent in situ flushing with Celsior solution (IGL) and static preservation in an ice-box.Liver MP was applied to all DCD grafts and to DBD extended-criteria donor (ECD) organs showing one of the following characteristics: expected prolonged ischemia time (>10 h), macrovescicular steatosis >30%, or serum levels of hepatonecrosis markers exceeding 4 times the reference range.2 = 100%) at a temperature of 8–10 °C.MP was performed as back-to-base, end-ischemic dual hypothermic oxygenated machine perfusion (D-HOPE). Briefly, D-HOPE was performed using the Liver Assist device , and consisted in a double perfusion (4000 mL of circulating Belzer MPS-UW Machine Perfusion) through the portal vein and hepatic artery with pressures set at 4 and 25 mmHg, respectively. Grafts were actively oxygenated (FiOLiver transplantation (LT) was performed with a modified piggyback technique and bile duct anastomoses over a T-tube. Post-LT course and biochemical analysis were performed .Early allograft dysfunction (EAD) was defined as: peak transaminases > 2000 IU/L (within the first week after LT), international normalized ratio > 1.7, or bilirubin > 10 mg/dL on postoperative day (POD) 7 . PrimarySamples of effluent were processed to obtain cell-free supernatants and donor-derived leukocyte pellets . A biochemical panel including transaminases , Blood Urea Nitrogen (BUN), glucose, lactate, LDH, D-dimer, and electrolytes was assessed following standard techniques.The FMN concentration was determined by fluorescence spectroscopy. Briefly, 5 mg of Riboflavin 5′-monophosphate sodium salt hydrate  were dissolved in either 0.9% sodium chloride or Celsior. Thereafter, these solutions were serially diluted to obtain different standard FMN solutions with concentration ranging from 31.25 to 1000.00 ng/mL . EffluenHepatocyte apoptosis was assessed by measuring the release of caspase-cleaved CK18 with a commercially available immunoassay based on the use of M30 monoclonal antibody that specifically detects the neoepitope ccK18/K18-Asp396 . The analysis was performed following manufacturers’ instructions and the absorbance was determined using a microplate reader (Synergy HTX).® Technology  or commercial available enzyme-linked immunosorbent assay (ELISA) assays . A list of all the measured analytes with low and high standards as well as test sensitivity is provided in the A panel of soluble proteins relevant to inflammation and its resolution , glycocalyx , coagulation  was evaluated using custom-designed immunoassays based on Luminex xMAPCell pellets were suspended in 0.25 mL 1X PBS solution (Sigma-Aldrich) and counted using an automated cell counter .Free hemoglobin concentration was assessed by applying the Allen correction to absorbance readings at 563 nm, 577 nm, 600 nm (Synergy HTX).Statistical analysis was performed using the JMP Pro 15 software . Box plots and linear regression plots were created using SigmaPlot 11.0 .Continuous variables are presented as median  grafts were subjected to MP, where 10 were procured from DCD and 8 from DBD-ECD donors meeting the criteria specified above, except for livers 18 and 19, which were subjected to MP to extend the preservation time because of logistic reasons (change of recipient) ,45,52.The mean recipient model for end stage liver disease (MELD) score was 13 (9–17) points, while 11% of the patients received an urgent allocated graft . Pre-LT Despite an overall high-risk population in our cohort, graft survival, censored for tumor-related death, remained comparable to the levels seen in an ideal LT cohort presented in a recent benchmark study (p < 0.05): (1) TNF-α, IL-1β, CXCL8/IL-8, CXCL9/MIG; (2) ANGPTL3, ANGPTL4, Galectin-9, HGF; (3) AST, ALT, LDH.Different correlations across the variables assessed in effluent fluids were found through multivariate analysis . The folp = 0.040), ANGPTL3 , ANGPTL4 , and CXCL9 . HA was associated with both inflammatory mediators  and markers of hepatocyte injury . The apoptosis marker CK18 correlated with CCL2 , HGF , IL-6 , and free hemoglobin . Finally, a strong association was observed between FMN and free hemoglobin .Of note, IL-10 was inversely associated with TNF-α . Out of the nine grafts excluded from the “MP-yes” cluster, six were procured from DCD donors , while three were from DBD donors . Of note, three livers were grouped in a distinctive sub-cluster due to high expression of inflammation-related molecules (p = 0.018).Unsupervised clustering analysis disclosed a distinctive signature in the bio-molecular profile of livers referred to MP procedure. In fact, half of the grafts subjected to MP  were grouped together due to their similar profiles (p = 0.419), ALT (0.22 (0.20–0.38) IU/g vs. 0.42 (0.18–0.70) IU/g, p = 0.260), and LDH (0.52 (0.35–0.86) IU/g vs. 0.64 (0.40–1.34) IU/g, p = 0.437).Consistently, statistical analysis indicated substantial differences between effluents collected from livers referred to MP compared to those of the grafts immediately used for transplantation. In particular, the “MP-yes” group was associated with increased effluent concentrations of the inflammatory molecules PTX3, CXCL8/IL-8, TNF-α, and ANGPTL4 . ConversData reported in Finally, no relationship was found between the concentration of lactate, AST, and ALT assessed in NRP perfusates and the release of these factors in effluent fluids.p = 0.016). With regard to the other biomarkers assessed in effluent fluids, no significant difference was observed between the “EAD-yes” and the “EAD-no” groups , recipient serum AST , and ICU stay . Of interest, strong or moderate correlations were found between effluent HA and recipient creatinine, AST, and ICU stay (p = 0.009) and CXCL8 .A further analysis investigated any potential association between the variables measured in the effluent fluids and clinical data related to transplantation outcome, including days of recipient hospitalization in intensive care unit (ICU) and biochemical parameters of liver and kidney function assessed in recipients’ plasma samples (peak). Effluent biomarkers of liver damage correlated with recipient serum creatinine  cells, and lymphocytes . Of noteWith regard to traditional markers of liver injury, there was no significant difference in effluent concentration of AST, ALT, and LDH of livers referred to MP, compared to grafts not subjected to ex situ perfusion. Of note, the present study includes the evaluation of FMN, a novel biomarker of cell viability, whose concentration is measured in perfusate samples to assess liver graft mitochondrial function throughout HOPE . AlthougAdditional analysis documented a peculiar inflammation, cell death, and coagulation profile in fluids collected from DCD livers, compared to those obtained from DBD grafts. This condition could reflect the detrimental influence of prolonged WIT secondary to the 20 min no-touch period, which is mandatory in the Italian setting . MoreoveFinally, correlations between recipient short-term outcome and effluent concentration of either cytolysis biomarkers or HA were observed. Along with biliary complications and AKI, EAD is a post-LT complication  mainly cA significant limitation in the present study resides in the small number of liver transplantations performed in our center, which limits the validation of each of the variables assessed in effluent fluids. In addition, based on the excellent outcomes with a low number of complications, despite the high donor risk, we were not able to correlate effluent molecular analysis with recipient outcomes. Targeted studies are required to better characterize the prognostic ability of effluent biomarkers to predict the occurrence of post-transplant complications.The present proof-of-concept study shows that molecular analysis of effluent samples can provide objective criteria reflecting the cumulative effects of noxious stimuli affecting liver graft quality during the organ donation process. We suggest that bio-molecular stratification of livers would significantly contribute to implement the clinical strategy with or without application of the MP procedure, leading to improved, cost-saving, and tailored healthcare resource utilization based on the specific injury experienced by each graft. In addition, marginal organs that are currently deemed unsuitable because of the presumed high-risk to develop complications could be successfully used in case a “healthy” biomarker profile is found. Of interest, novel technologies can enable surgeons to perform real-time evaluation of effluent fluids during back table preparation ,69, with


Anterior cruciate ligament (ACL) tear is common in sports and accidents, and accounts for over 50% of all knee injuries. ACL reconstruction (ACLR) is commonly indicated to restore the knee stability, prevent anterior–posterior translation, and reduce the risk of developing post-traumatic osteoarthritis. However, the outcome of biological graft healing is not satisfactory with graft failure after ACLR. Tendon graft-to-bone tunnel healing and graft mid-substance remodeling are two key challenges of biological graft healing after ACLR. Mounting evidence supports excessive inflammation due to ACL injury and ACLR, and tendon graft-to-bone tunnel motion negatively influences these two key processes. To tackle the problem of biological graft healing, we believe that an inductive approach should be adopted, starting from the endpoint that we expected after ACLR, even though the results may not be achievable at present, followed by developing clinically practical strategies to achieve this ultimate goal. We believe that mineralization of tunnel graft and ligamentization of graft mid-substance to restore the ultrastructure and anatomy of the original ACL are the ultimate targets of ACLR. Hence, strategies that are osteoinductive, angiogenic, or anti-inflammatory should drive graft healing toward the targets. This paper reviews pre-clinical and clinical literature supporting this claim and the role of inflammation in negatively influencing graft healing. The practical considerations when developing a biological therapy to promote ACLR for future clinical translation are also discussed. The anterior cruciate ligament (ACL) is a band of dense connective tissues that courses from the femur to the tibia, the function of which is to prevent excessive knee anterior–posterior translation and to maintain joint stability . Tears oACL tears can impair knee function and increase the lifetime risk of knee osteoarthritis. Patients without and with meniscal tear had a 0%–13% and 21%–48% higher risk of developing knee osteoarthritis at 10 years after an ACL injury, respectively . The conACL tears are managed by conservative and surgical approaches. Cryotherapy, restrictive bracing, continuous passive motion, electrotherapy, and exercises aimed at reducing inflammation, restricting excessive knee motions, or strengthening muscles to improve knee symptoms and stability as well as to protect the joint are common conservative treatment options. However, conservative approaches for the management of ACL tears are poorly accepted by young active individuals. The continued active lifestyles of these individuals often lead to recurrent knee instability, chondral and meniscal injuries, and early onset of osteoarthritis .ACL reconstruction is a surgical procedure that replaces the injured ACL with a tendon graft as the ACL does not heal after injury due to insufficient vascularization. Bone tunnels are artificially created in the distal femur and proximal tibia, and the tendon graft is inserted and fixed to the bone tunnels using staples, sutures, cross-pins, or interference screws. The procedure is usually assisted by arthroscopy to minimize the size of incision and reduce complications. The success rate of ACLR varied from 73% to 95% and the return to pre-injury activity level varied from 37% to 75% . Graft fTo tackle the problem of poor graft healing after ACLR, we believe a new research paradigm should be applied. An inductive approach, starting from the ideal endpoint of ACLR, should be adopted. The feasibility of translating the pre-clinical findings into clinical practice should be considered at the pre-clinical stage. This would enhance the future translation of the findings to benefit patients. Using this new paradigm of thinking, we believe that strategies that are osteoinductive, angiogenic, or anti-inflammatory should drive graft healing toward the ultimate goal of restoring ACL function after ACLR. This paper aims to present evidence from the literature to support this claim. The graft healing process is first summarized, with evidence supporting the role of inflammation in negatively influencing the healing response highlighted. Evidence supporting osteoinductive, angiogenic, or anti-inflammatory approaches to promote graft healing will then be systematically reviewed. Important issues requiring consideration when developing biological therapies for ACLR from the perspective of future clinical translation are then discussed.Tendon graft-to-bone tunnel healing followed by graft mid-substance remodeling  are two key healing processes that occur after ACLR . After ATendon graft-to-bone tunnel healing is the weak link in the early stage of ACLR as it requires the attachment of a compliant material-like tendon to a relatively stiff material-like bone. The regeneration of a normal insertion site with formation of a unique transitional tissue called “enthesis” , charactDepending on how the collagen fibers are attached to bone, there are two types of entheses at the bone–tendon junction—direct and indirect insertions. Direct insertion  is composed of four zones  in orderAs healing progresses, the weak link of graft healing gradually shifts from the tendon graft-to-bone tunnel interface to the graft mid-substance. The tendon graft undergoes “ligamentization,” in which the composition and organization of ECM are adapted to the functions of an active ACL.Ligament fibroblasts have higher DNA content and are more metabolically active compared with tendon fibroblasts. Besides, the ECM of ligament contains more type III collagen and glycosaminoglycans but slightly less total collagen compared with tendons. Due to the direction of mechanical load, the collagen fibers of ligament are less orderly aligned compared with tendon. Moreover, the collagen fibrils in ligament are smaller but have more reducible cross-links compared with the fibrils in tendon .The process of ligamentization, therefore, requires the tendon graft to metabolically, compositionally, and ultrastructurally remodeled to adapt to the function of a ligament. However, the process is not successful as graft degeneration due to poor angiogenesis and ECM degradation were observed both clinically  and in aShortly after graft implantation, an inflammatory response, which is important for removing cell debris and activate tissue repair, ensues. Neutrophils and macrophages are recruited to the tendon–bone interface as early as 4 days after surgery . They prTo confirm the association of inflammation with poor outcome of ACL graft healing, a systematic search and analysis of the literature was done  , α-fibro+ cell , tendon-+ cell , bone ma+ cell , and adi+ cell  had been+ cell  to lengt+ cell  containiClinically, platelet-rich plasma (PRP) containing osteogenic factors is frequently tested for the promotion of graft healing after ACLR with mixed results. It has been reported to prevent tunnel widening  and reduIn summary, all pre-clinical studies have shown positive effects of osteoinductive factors on the promotion of bone formation and enhancement of tendon graft-to-bone tunnel healing, whereas the graft healing effect of PRP, which contain a complex mixture of bioactive proteins other than osteoinductive factors, remains controversial. More well-controlled studies are needed to confirm the effect of PRP on osteogenesis and graft healing after ACLR.Adequate revascularization is critical for successful tendon graft-to-bone tunnel healing, by transporting MSCs and growth factors to the injury site. The infiltrated MSCs repopulate the tendon graft, release growth factors, produce collagen, and differentiated into cells of different lineages  . Vascula+ cell sheet (The administration of angiogenic factors such as platelet-derived growth factor-BB (PDGF-BB) , VEGF S, and α-fll sheet , or BMSCll sheet  containill sheet . Tight cThere has been a long-term interest in the application of PRP and its related-blood preparations for the promotion of graft healing due to its angiogenic components . The admAs discussed, inflammatory cytokines and fibrogenic growth factors contribute to the formation of a fibrous tissue interface between the graft and the bone tunnel at the early healing phase . While tIn our systematic search for biological interventions, only one preclinical study and one clinical study evaluating the efficacy of anti-inflammatory therapies could be identified. TGF-β1 is an anti-inflammatory growth factor. Kondo et al. transplanted TGF-β1-pretreated synovium-derived cells or TGF-β1 in a fibrin sealant sheet to a sheep ACLR model and found that both the pre-treated cells and TGF-β1 inhibited the deterioration of the intra-articular part of tendon graft with increased maximum load and stiffness of the bone–graft–bone complex at 12 weeks after reconstruction. Similar results were not observed after the transplantation of untreated cells in fibrin sealant sheet, suggesting that TGF-β1-inhibited graft degeneration is crucial for graft healing , showingRole of inflammation in influencing healing response section), there has been relatively few original studies targeting inflammation in graft healing after ACLR compared with the number of studies investigating biological therapies targeting osteogenesis and angiogenesis. Improved understanding of the molecular mechanisms of inflammatory cells and cytokines in graft healing after ACLR would support the identification of new anti-inflammatory therapeutics.Despite the existence of clear evidence demonstrating the association of inflammation with poor healing outcomes (While pre-clinical studies have shown positive effects of various biological therapies on graft healing, additional factors need to be considered for translating them into clinical practices. We suggested considering the following factors when developing new biologics at the early pre-clinical stage for generating clinically viable treatment strategies.Arthroscopy is a minimally invasive surgical procedure on a joint in which an arthroscope is inserted into the joint through a small incision to assist in knee examination and surgery. It is a common orthopedic procedure, and about 60,000 arthroscopic surgeries are performed annually in England only . For artAs tendon graft-to-bone tunnel healing and graft mid-substance remodeling are complex healing processes with different endpoints , localizVarious sources of cells have been used for ACLR . Issues PRP is another complex biological product that has been widely studied for the promotion of graft healing in ACLR both in preclinical and clinical studies with mixed results . There aExcept pure proteins, biological therapies are usually complex products. In order to ensure their safety and efficacy in graft healing after ACLR, quality control is required. The adoption of an inductive approach in the research and development of new biologics for improving graft healing after ACLR with the consideration of the feasibility of clinical translation would help in refining the scope of the clinical problem and directing the search for the right strategies.The bone tunnels in ACLR are artificially created. The ultimate targets after ACLR are to replace the tunnel graft with bone with the re-establishment of a direct insertion at the original ACL footprints and remodeling of graft mid-substance to a functional ACL. However, the remodeling of graft mid-substance always fails due to its avascularity. In addition, there is amble evidence showing that excessive inflammation due to an increase in inflammatory cytokines in the synovial fluid after ACL injury, surgical trauma in ACLR, and graft-to-bone tunnel motion because of unstable fixation and slow healing contribute to tunnel widening, bone resorption, and graft mid-substance degeneration. Therefore, strategies that can promote osteogenesis, angiogenesis, and anti-inflammation have a good chance of enhancing graft healing. This claim is supported by the detrimental effects of excessive inflammation on the healing outcomes, and the osteogenic, angiogenetic, or anti-inflammatory properties of most of the biologics that showed positive effects on graft healing in a systematic literature search. To enhance the translational potential of a new biologic for graft healing, its compatibility with the standard ACL surgical procedures, such as arthroscopy, its localized and sustained release pattern, and its quality control, are key issues to be considered as early as at the pre-clinical stage.