Dairy cow longevity is a major concern in most high milk-producing countries. Failure to reproduce is the most common reason for involuntary culling worldwide. A high prevalence of metabolic and infectious diseases (DIS) in dairy herds is not unusual; however, the effects of DIS on cow fertility vary and the reason for this variation is currently unclear. One and a half months after parturition, approximately 30% of cows experience uterine inflammation characterised by an increased proportion of white blood cells, more specifically polymorphonuclear neutrophils in the uterine lumen—a disease termed cytological endometritis (CYTO). CYTO is associated with a decreased conception rate and higher culling risk. Accordingly, we studied the influence of CYTO on the fertility and culling of clinically healthy and unhealthy dairy cows. We found that CYTO+ status, irrespective of DIS status, decreased the pregnancy rate. When DIS+ and CYTO+ were combined, they had an additive negative effect. This finding led us to conclude that the variation in the effects of clinical disease on reproduction can at least partly be explained by the occurrence of CYTO. Therefore, in dairy herds with fertility problems, the prevalence of CYTO should be assessed. Further investigations including a larger number of cows are required to confirm these results.The objective of this study was to evaluate the effect of calving-related disorders/clinical diseases (DIS) and cytological endometritis (CYTO) on the reproductive performance and culling of dairy cows. In a total of 119 multiparous Holstein cows, DIS were recorded and CYTO was diagnosed at 40 ± 2 d in milk. Onset of luteal activity was defined as the first postpartum measurement of milk progesterone >5 ng/mL. A dummy variable containing all four possible permutations between DIS and CYTO statuses was created. The pregnancy rates after first artificial insemination were 57.7, 42.9, 23.0 and 15.8% for the DIS−/CYTO−, DIS+/CYTO−, DIS−/CYTO+ and DIS+/CYTO+ groups, respectively. The hazard of pregnancy was affected by DIS−/CYTO+ and DIS+/CYTO+ health statuses (hazard ratio (HR) = 0.43 and 0.29, respectively), whereas DIS+/CYTO− and DIS−/CYTO− cows had a similar hazard to pregnancy. The hazard of onset of luteal activity was affected by DIS+/CYTO+ health status (HR = 0.45), but not by DIS+/CYTO− and DIS−/CYTO+ health statuses. Compared with DIS−/CYTO− cows, DIS−/CYTO+ and DIS+/CYTO+ cows had 4.24 and 5.57 times the odds of being culled, respectively. Culling was not affected by DIS+/CYTO− health status. In conclusion, CYTO+ status, irrespective of DIS status, decreased the pregnancy rate. When DIS+ and CYTO+ were combined, they had an additive negative effect. Our preliminary findings suggest that the far-reaching consequences of clinical diseases on fertility and culling can be mediated through the development of CYTO. Further investigations including a larger number of cows are required to confirm these results. Dairy cow longevity has decreased over the years in most high milk-producing countries, with failure to reproduce being the most common reason for involuntary culling worldwide . Poor reDuring the periparturient transition period, dairy cows are subjected to a variety of metabolic and physiological changes that affect productivity, health and future reproductive performance. Despite tight homeostatic controls and homeorhetic adjustments to cope with the changes in metabolism, a high prevalence of calving-related disorders and metabolic or infectious diseases is not uncommon, with 45–71% of dairy cows across different levels of milk production, breeds and management systems affected ,4,5,6,7.Fourichon et al.  with datCytological endometritis (CYTO) is a postpartum uterine inflammation characterised by an increased percentage of inflammatory cells, principally polymorphonuclear neutrophils (PMN) in endometrial cytology samples, which results in a significant reduction in reproductive performance ,11. CYTOAt present, there are no data on the combined effects of calving-related disorders/clinical diseases and CYTO on the reproductive performance and culling of dairy cows. Previous investigations ,21,22,23The present study is the first of its kind in that it evaluates the individual and combined effects of diseases and CYTO on the reproductive performance and culling of dairy cows. Therefore, the central hypothesis of this study is that the variance in the effect of clinical diseases on reproduction and culling is related to the CYTO status of the animals. Specifically, unhealthy cows with a CYTO-positive diagnosis resume postpartum luteal activity later, suffer greater detrimental effects concerning fertility and have a higher culling risk compared to unhealthy cows with CYTO-negative health status.Because an increase in parity is associated with inferior reproductive performance ,25,26 anThe preliminary findings presented here suggest that the far-reaching consequences of clinical diseases in early postpartum on future fertility and culling can be mediated through the development of CYTO.ad libitum intake. Cows were milked twice daily with parallel rotary milking parlour  and daily milk yields were measured with flow milk meters . The cows were selected for AI on spontaneous oestrus, detected by collar mounted activity meters  combined with visual observation for standing to be mounted and secondary oestrous signs. Cows were inseminated with frozen-thawed semen of proven Holstein sires after a voluntary waiting period of 60 days. One experienced herd AI technician conducted all the inseminations. The AI technician was blinded to cow history and health status. Pregnancies were diagnosed from milk progesterone (P4) profiles and confirmed by rectal palpation at 65 to 70 days after AI. The cows were vaccinated against bovine viral diarrhea and infectious bovine rhinotracheitis viruses.This was a retrospective cohort study. A description of the studied animals has previously been reported . BrieflyCalving-related conditions and clinical diseases were diagnosed, confirmed, documented and treated by an experienced farm veterinarian. There was no indication of presence of infectious diseases. The conditions and diseases diagnosed included twinning, dystocia, retained placenta, metritis, clinical endometritis, clinical hypocalcaemia, ketosis, clinical mastitis and severe lameness. Dystocia was defined as providing assistance without or with the use of mechanical traction. Retention of foetal membranes was characterised as the presence of foetal membranes at least 12 h after calving. Metritis (watery red-brown fluid to viscous off-white purulent uterine discharge independent of fever before 21 days in milk (DIM)) and clinical endometritis  were defined according to Sheldon et al. . Clinica®; Medscand Medical, Malmö, Sweden) attached to a stainless-steel device for use in cows (European Patent EP2029026B1). Before use, the devices were mounted with Uterobrush®, individually packed in 75-mm Steriking® transparent heat-sealable roll  and sterilised by autoclaving. After thorough cleaning of the vulva, endometrial cytology samples were taken from the base of the larger uterine horn. Under guidance per rectum, the device was introduced into the desired region of the uterus, the rod-fixing screw was loosened and the rod of the device was pushed into the guiding tube. As a result, the Uterobrush® advanced into the uterus. By rotating the rod multiple times around its axle, endometrium material was collected on the Uterobrush®. The Uterobrush® was then retracted into the device, the rod was fixed with the aid of the rod-fixing screw and the device was taken out of the uterus. Between uses, the devices were washed, equipped with new Uterobrush®, individually packed and autoclaved. All cytology samples were taken by the same researcher. Slides for cytologic examination were prepared by rolling the Uterobrush® onto a clean glass microscope slide and immediately fixing in a current of warm air using a blow drier. Four slides from each cow were prepared. The slides were then transferred to the laboratory and two of the four slides from each cow were stained with May-Grünwald Giemsa procedure and examined by light microscopy under 400× and 1000× magnification . For each slide, surveying 100 cells, the numbers of epithelial cells and PMN were counted and the percentage of PMN calculated as described by Kasimanickam et al. [Endometrial samples for cytological examination were collected at 40 ± 2 (mean ± SD) days after calving using an endometrial brush  at week +3 postpartum using a 1 (emaciated) to 5 (obese) scale with 0.25-unit increments according to Ferguson et al. . Cows wiLocomotion was scored every fortnight on a 5-point scale in increments of 1 using the method described by Sprecher et al. . Cows wiTo evaluate the individual and combined effects of calving-related conditions/clinical diseases (DIS) and CYTO, a dummy variable containing all four possible permutations between health and CYTO statuses was created: condition/disease-negative—CYTO−negative (DIS−/CYTO−); condition/disease-positive—CYTO-negative (DIS+/CYTO−); condition/disease-negative—CYTO-positive (DIS−/CYTO+); and condition/disease-positive—CYTO-positive (DIS+/CYTO+).Parity was created as a binary variable including lactation number 2 and >2.To determine the distribution of different clinical diseases between DIS+/CYTO+ and DIS+/CYTO− cows, the DIS+ cows were grouped into three categories as follows: (1) cows that had clinical uterine inflammatory disease only, which included purulent vaginal discharge ; (2) cows that had clinical uterine inflammatory disease and were also affected by another calving-related condition/disease; and (3) cows that had only non-uterine diseases, which included clinical mastitis, clinical hypocalcaemia and severe lameness.The effects of health and CYTO statuses (DIS/CYTO group) on pregnancy to first service and culling were analysed by logistic regression. The effects of DIS/CYTO group on onset of luteal activity and time to pregnancy were determined by Cox’s proportional hazard models. The survival time was right censored at 255 DIM or at the date of culling if culling occurred before 255 DIM. The proportional hazards assumption was tested in R using statistical software R 3.3.3  using cox.zph in the survival package .p-values were Bonferroni corrected. Shapiro-Wilk test was used to analyse the distribution of residuals.First 45-day cumulative milk yields among the four DIS/CYTO groups were compared by ANCOVA. p < 0.2), submitted to the multivariable model(s). The covariates were retained in the multivariable model(s) if associated with outcome (p < 0.15).The potential confounding effects of parity , BCS at week +3 postpartum  and time from calving to first AI were tested univariately and, if associated with outcome  was used for statistical analyses and preparation of survival curves. Figures were prepared using Prism 8 for Windows V.8.4.3 .MedCalcDescriptive statistics of the measured parameters in the studied cows are given in The proportions of cows affected by twinning, retained placenta, metritis/purulent vaginal discharge, clinical mastitis, clinical hypocalcaemia, severe lameness and CYTO were 6.7, 5.9, 30.3, 7.6, 1.7, 5.9 and 30.3%, respectively. In the 119 studied cows, 20 (16.8%) had more than one health disorder/problem and 68 (57.1%) did not have any calving complication or health disorder/problem. The proportions of DIS−/CYTO−, DIS+/CYTO−, DIS−/CYTO+ and DIS+/CYTO+ cows were 45.4 (54/119), 24.4 (29/119), 11.8 (14/119) and 18.5% (22/119), respectively.p = 0.004) and culling (p = 0.010). The proportions of cows pregnant after first AI were 57.7, 42.9, 23.0 and 15.8% and the proportions of cows culled were 7.4, 10.3, 28.6 and 36.4% for DIS−/CYTO−, DIS+/CYTO−, DIS−/CYTO+ and DIS+/CYTO+ cows, respectively. The logistic regression model (p = 0.210) than DIS−/CYTO+  and DIS+/CYTO+ cows . Accounting for the effect of parity (p = 0.14), the odds of culling in DIS+/CYTO− cows did not differ from that in DIS−/CYTO− cows . DIS−/CYTO+ cows had 4.24 times the odds (p = 0.072) and DIS+/CYTO+ cows had 5.57 times the odds (p = 0.015) of being culled compared with DIS−/CYTO− cows.DIS/CYTO group affected pregnancy after first AI , DIS+/CYTO+ cows took a longer time to resume cyclicity (hazard ratio (HR) = 0.45, p = 0.004), whereas DIS+/CYTO− cows and DIS−/CYTO+ cows had a similar hazard to resume cyclicity  and HR = 1.09, p = 0.772, respectively) when compared to DIS−/CYTO− cows (p = 0.58) and was not incorporated into the model.Accounting for BCS at week +3 postpartum effects (TO− cows . The intp = 0.121) and time to first AI (p = 0.007) effects, DIS−/CYTO+ and DIS+/CYTO+ cows took a longer time to pregnancy , whereas DIS+/CYTO− cows had a similar hazard to pregnancy  when compared to DIS−/CYTO− cows  of being pregnant at first service when compared to DIS+/CYTO− cows. After adjusting for lactation number (=2 and >2), DIS+/CYTO+ cows had 4.91 times the odds  of being culled when compared to DIS+/CYTO− cows.CYTO was a clinical disease effect modifier. The logistic regression models revealed that unhealthy cows with a negative CYTO diagnosis had superior fertility and lower culling risk when compared to unhealthy cows with a positive CYTO diagnosis. Specifically, DIS+/CYTO+ cows had 0.25 times the odds  when compared to DIS+/CYTO− cows. After adjusting for time to first AI and BCS at week +3 postpartum (≥2.75 and ≤2.5), the Cox’s proportional hazard model showed that DIS+/CYTO+ cows were 59% less likely to become pregnant  when compared to DIS+/CYTO− cows.After adjusting for BCS at week +3 postpartum (≥2.75 and ≤2.5), the Cox’s proportional hazard model showed that DIS+/CYTO+ cows were 55% less likely to resume cyclicity  between the DIS+/CYTO+ and DIS+/CYTO− cow groups (p = 0.009).The proportions of cows with non-uterine disease only, cows with metritis/purulent vaginal discharge only and cows with metritis/purulent vaginal discharge plus another complication/disease did not differ (w groups . Howeverp < 0.001). The first 45-day cumulative milk yield (mean ± SD) was lower in DIS+/CYTO− (1354.9 ± 286.15 kg) and DIS+/CYTO+ (1221.5 ± 442.13 kg) cows compared with DIS−/CYTO− (1564.9 ± 226.09 kg) and DIS−/CYTO+ (1668.0 ± 341.73 kg) cows. This variable did not differ between the DIS−/CYTO+ and DIS−/CYTO− cow groups . Interestingly, there was no difference in the proportion of cows with uterine and non-uterine diseases between the DIS+/CYTO+ and DIS+/CYTO− cow groups (p = 0.616)  . Out of Individual cows kept under identical conditions show different adaptations to metabolic stress . The samRecent studies strongly suggest that endotoxins play a significant role in the development of multiple periparturient diseases currently affecting transition dairy cows ,53. The p < 0.001). In line with the study of Manríquez et al. [p = 0.750). Manríquez et al. [Endotoxins are able to disrupt the hypothalamus-pituitary-ovary axis, thus affecting gonadotropin-releasing hormone and preovulatory luteinizing hormone release ,64, follz et al. , there wz et al.  studied z et al. , the avaz et al. ,73.This is the first study to demonstrate the additive effects of clinical disease and CYTO on postpartum onset of luteal activity. Previous studies have presented inconsistent results regarding the effect of CYTO on the interval from calving to first ovulation. In agreement with the findings of Senosy et al. , we founBoth the DIS−/CYTO+ and DIS+/CYTO+ cow groups had reduced fertility and higher culling risk . An inflThe drastically reduced fertility and high culling risk in cows with both clinical disease and CYTO (DIS+/CYTO+ group) may be a combination of effects of compromised follicular growth development, delayed start of ovarian cyclicity, compromised oocyte developmental competence and survival, reduced sperm motility, fertilization failure, low embryo quality and impaired uterine ability to support implantation and pregnancy.Removal of cows from the herd was largely explained by those that had both clinical disease and CYTO or that were clinically healthy but had CYTO. The odds of removal from the herd were 5.6- and 4.2-fold greater for DIS+/CYTO+ and DIS−/CYTO+ cows, respectively, compared with DIS−/CYTO− cows. Culling was not affected by DIS+/CYTO− health status, indicating that despite DIS+/CYTO− cows having postpartum problems and/or diseases, they were still able to regain homeostasis, resume cyclicity and become pregnant. Getting pregnant explains the similar culling risk for DIS+/CYTO− and DIS−/CYTO− cows. The identification of single and combined effects as potential predictors of impaired reproduction and increased culling of cows is highly beneficial for the goal of improving the management and profitability of dairy herds .p = 1.0). However, DIS−/CYTO+ cows had a lower first AI pregnancy rate than DIS−/CYTO− cows  and were 57% (p = 0.019) less likely to become pregnant. Published data on the association of milk production with fertility are conflicting [An interesting finding was that milk yield during the first 45 days of lactation in DIS−/CYTO+ cows did not differ from DIS−/CYTO− cows  did not affect onset of luteal activity. Our data suggest that the far-reaching consequences of clinical diseases on fertility and culling can be mediated through the development of CYTO. In herds with fertility problems, special attention needs to be paid to the occurrence of CYTO, both in cows with calving-related disorders and/or clinical diseases and in clinically healthy cows.This study is a proof of concept; future studies with larger number of animals are needed to confirm our findings and to further evaluate the effect of calving-related conditions/clinical diseases and the effect of CYTO on the fertility and culling of cows belonging to different breeds that are kept in different environmental and managemental conditions. Furthermore, the mechanisms underlying why some cows with or without clinical evidence of disease, in similar environmental conditions, develop CYTO and others do not need to be established.


Klebsiella pneumoniae strains isolated from clinical cases in Tehran, Iran. A total of 100 different types of clinical human samples were collected from a major teaching hospital in Tehran, Iran. Bacterial isolates were identified using standard microbiological tests. Antimicrobial susceptibility testing was done according to the latest CLSI guidelines. PCR was used to amplify the gyrA gene in quinolone-resistant isolates and sequencing was performed for the detection of probable mutations between the isolates. The occurrence of plasmid-mediated quinolone resistance genes  was also investigated by PCR. Finally, genotyping of the strains was performed by PFGE in a standard condition. The susceptibility pattern revealed a high and low level of resistance against meropenem (20%) and trimethoprim (37%), respectively. PCR and sequencing detected mutation in the gyrA gene in 51% of quinolone-resistant K. pneumoniae. According to the susceptibility report, among nalidixic acid-resistant strains, 60.5%, 50%, and 42.9% of isolates contained qnrA, qnrB, and qnrS, respectively. Among ciprofloxacin-resistant strains, qnrA was the most frequent PMQR gene. The PFGE differentiated the strains into 31 different genetic clusters so that the highest number (7/66) was in category A. Our results indicated that the frequency of resistance to various antibiotics particularly trimethoprim, nalidixic acid, and cefoxitin are increasing. The presence of qnr (S and A) genes and point mutation of the gyrA gene were likely to be responsible for the resistance toward nalidixic acid and ciprofloxacin in our strains. Also, the results obtained from genotyping indicated that the K. pneumoniae strains isolated in this study belonged to the diverse clones.The aims of this study were the molecular characterization of antibiotic resistance and genotyping of  Klebsiella pneumoniae is one of the most common pathogens in hospitals with high mortality rates and causes a variety of infections including pneumonia, urinary tract infections, septicemia, diarrhea, liver abscess, endophthalmitis, meningitis, and bacteremia [cteremia , 2.K. pneumoniae isolates is one of the serious problems of antibiotic treatment and causes a lot of worries in the world [K. pneumoniae infections more efficiently. The qnr genes are one of the plasmid-mediated quinolone resistance agents, which cause very rapid resistance to Enterobacteriaceae due to their placement on different integrons [qnr-mediated resistance among fluoroquinolones and quinolones-resistant Enterobacteriaceae isolates have been reported, making the treatment of these strains more complicated [The rapid emergence of drug resistance to he world . The avantegrons –6. Quinontegrons . In receplicated .K. pneumoniae strains isolated from clinical specimens in a major referral hospital in Tehran, Iran.Molecular typing is an important infection control tool to evaluate relationships between different isolates of bacteria. It helps us determine the sources of contamination, investigate the distribution status of pathogens, know how many pathogens changed over time, choose the best treatment of diseases, and reduce the risks of antibiotic resistance . The earK. pneumoniae by standard microbiological and biochemical methods [16SrRNA gene [During one year, a total of 100 clinical human samples including urine, blood, and sputum were collected from a major referral hospital in Tehran , Iran. Midstream urine was collected in sterile condition to decrease potential bacterial, cellular, and artifactual contamination. The bacterial isolates were identified as  methods . In addiRNA gene , 12.K. pneumoniae isolates was performed according to the Kirby–Bauer disk diffusion method. The Mueller–Hinton agar  medium was used for this purpose. Principles of the Clinical and Laboratory Standards Institute (CLSI) guidelines were used for this purpose to assess antibiotic resistance using trimethoprim (5 μg), cefoxitin (30 μg), ciprofloxacin (5 μg), nalidixic acid (30 μg), nitrofurantoin (300 μg), tetracycline (30 μg), streptomycin (10 μg), gentamicin (10 μg) and meropenem (10 μg) [K. pneumoniae ATCC 4352 was used as a quality control organism [Antibiotic susceptibility testing of  (10 μg) . All of organism .K. pneumoniae were extracted using a DNA extraction kit  according to the manufacturer's instructions. PCR was used to amplify the gyrA gene in nalidixic acid and ciprofloxacin-resistant strains as described previously [gyrA gene among the isolates.The DNAs of eviously . SequencqnrA, qnrB, and qnrS) were amplified using the primer sets listed in μl of each primer, 2.5 μl PCR buffer (10x), 0.7 μl MgCl2 (50 mM), 0.7 μl of each 10 mM dNTP , 0.5 μl of 5 U Pfu DNA polymerase, and 1 μl of sample DNA. PCR assays consisted of initial denaturation at 94°C for 5 min, denaturation at 94°C for 1 min, annealing of primers at 51–53°C for 1 min, primer extension at 72°C for 1 min, and the final extension at 72°C for 7 min (w/v) agarose gels and visualized using an ultraviolet (UV) transilluminator .PMQR determinants  was used to digest genomic DNAs. All the PFGE steps were accomplished following the CDC-standardized procedure used by all PulseNet laboratories with some changes. The genetic relatedness of K. pneumoniae isolates was evaluated by PFGE analysis as previously described [The clonal relatedness of escribed .P value <0.05 was considered a statistically significant level.Statistics were subjected to Microsoft Office Excel . Statistical analysis was performed by means of the SPSS 21.0 statistical software . The chi-squared test and Fisher's exact two-tailed test were applied to measure any significant relationship. A K. pneumoniae and were subjected to the study. All positive strains were also approved using the 16SrRNA-based PCR amplification. Urine samples (56%) had the highest prevalence of K. pneumoniae, while blood (12%) had the lowest. A statistically significant difference was seen between the types of samples and the prevalence of K. pneumoniae (P < 0.05).A total of 100 hospital-acquired infections harbored K. pneumoniae strains isolated from different types of hospital-acquired infections. The antibiotic susceptibility pattern revealed a high level of resistance to trimethoprim (63%) followed by nalidixic acid (53%) and cefoxitin (45.45%), while the low level of resistance was against meropenem (20%), gentamycin (25%), and streptomycin (30%). A statistically significant difference was seen between the types of samples and the prevalence of antibiotic resistance (P < 0.05).gyrA gene in 51% of quinolone-resistant K. pneumoniae. The sequencing showed that the mutant isolates carried point mutations in the gyrA quinolone resistance determining regions (QRDR) at codon 83 or 87 which leads to the substitution of different amino acids in gyrA protein.Molecular analysis detected mutation in the qnrA, qnrB, and qnrS primers, respectively. According to the PCR results, 7%, 18%, and 38% of isolates contained qnrA, qnrB, and qnrS, primers respectively. A statistically significant difference was seen between types of samples and the prevalence of PMQR genes (P < 0.05). Moreover, 10% (10/100) of the isolates were found to be positive for both genes.The results of DNA amplification by the PCR method based on the primers described in qnrA, qnrB, and qnrS, respectively. While, 57.1%, 50%, and 39.5% harbored qnrA, qnrB, and qnrS, respectively. Moreover, among ciprofloxacin-resistant strains, qnrA was the most frequent PMQR gene, while, qnrS and qnrB were the most frequent PMQR genes among nalidixic acid susceptible strains. The qnrS and qnrA genes were found to be relatively higher among nalidixic acid and ciprofloxacin resistance isolates .From 100  Figures . The higsotype A . A statiK. pneumoniae is one of the most important opportunistic pathogenic bacteria in people with immunodeficiency and underlying diseases [qnr genes in this family in Iran has been limited to a few studies [diseases . The bacdiseases . Pathogediseases . In rece studies .K. pneumoniae isolates was related to trimethoprim (63%) followed by nalidixic acid (53%) and cefoxitin (45.45%) antibiotics. The lowest rate of resistance was seen toward meropenem (20%), gentamycin (25%), and streptomycin (30%). Recently in Iran, Dehghan et al. in 2016 examined the pattern of antibiotic resistance in 120 strains of K. pneumoniae isolated from urine specimens [In this study, the most antibiotic resistance in pecimens . The antpecimens .K. pneumoniae isolated from UTI in hospital and outpatients. Resistance patterns among their isolates were trimethoprim (61%), gentamycin (59%), nitrofurantoin (55%), nalidixic acid (72%), and ciprofloxacin (60%). With an exception of gentamicin and nitrofurantoin, their results are consistent with our findings [Also, in another study, Latifpour et al. in 2016 examined the prevalence and antibiotic resistance pattern of 150 strains of broad-spectrum beta-lactamases producing findings .K. pneumoniae for samples of UTI in a number of patients in intensive care. In this study, the antibiotic resistance of ciprofloxacin was 42.4%, nitrofurantoin was 38%, and gentamicin was 21.2% indicating the results of this study were not consistent with our finding [Ranjbaran et al. in 2013 evaluated the antibiotic susceptibility pattern of 50 isolates of  finding . The difgyrA gene showed the mutations in codons 83 and 87, resulting in the substitution of different amino acids in gyrA protein among the quinolone-resistant resistant K. pneumoniae. Previously, several studies in our country have shown this mutation can cause the replacement of the amino acid serine to phenylalanine (Ser83 ⟶ Phe), serine to tyrosine (Ser83 ⟶ Tyr), and serine to leucine (Ser83 ⟶ Leu) [The sequencing of the 3 ⟶ Leu) , 25.K. pneumoniae strains had the mutation of the gyrA gene [gyrA gene was reported among K. pneumoniae strains [gyrA among all quinolone-resistant K. pneumoniae isolates, followed by Asp87 ⟶ Asn [gyrA among 95% of ciprofloxacin-resistant Klebsiella strains isolated from China. Huang et al. in a recent study showed in [Kareem et al. reported that 40% of quinolone-resistant yrA gene . In anot strains . In Iraq87 ⟶ Asn . Huang ehowed in .qnr plasmid-mediated genes showed that among 100 isolates of K. pneumoniae 7%, 18%, and 38% of isolates contained qnrA, qnrB, and qnrS, respectively. A statistically significant difference was seen between types of samples and the prevalence of PMQR genes (P < 0.05). Moreover, 10% (10/100) of the isolates were found to be positive for both genes.The frequency of qnrA, qnrB, and qnrS genes among K. pneumoniae isolates obtained from hospitalized patients. The results obtained in that study showed that 52%, 22 25%, and 23% of isolates of K. pneumoniae contained the qnrB, qnrA, and qnrS genes, respectively [Salimizand et al., in 2021, studied the frequency of ectively .K. pneumoniae isolates in Iran. The results of their study showed that the most common qnr gene was qnrB (84.7%), followed by qnrS (65.2%) and qnrA (22.2%). The frequencies of qnr found in their study were higher than those obtained from our study [Taraghian et al. studied plasmid-mediated quinolone resistance in quinolone-resistant ur study .K. pneumoniae strains in Qatar. The rate of qnr A/B/E/S (plasmid gene was 45.3% in K. pneumoniae isolates) indicated the higher frequency of qnr plasmid genes compared to our study [Perez-Lopez et al., in 2020, examined plasmid-mediated quinolone resistance determinants in ur study .Therefore, findings suggest that the emergence of PMQR helps to rapidly increase the spread of bacterial resistance to fluoroquinolones, which requires continuous monitoring of antibiotic use .PFGE is one of the genotypic methods used for bacterial typing and is superior to other molecular methods due to its high differentiation in different bacterial strains. This method is able to differentiate the large genome of organisms . NowadayK. pneumoniae recovered from the patients with bacteremia and UTI in Mozambique [oqxA gene expression in 111 clinical isolates resistant to antibiotics of K. pneumoniae isolated from inpatients. They used the PFGE for genotyping resistant strains. Fifteen patterns of PFGE including 3 main clusters A, B, and C were identified [In this study, PFGE results showed 31 different genetic patterns among the strains that were arbitrarily named as A-AF. The highest number  was seenzambique . PFGE anentified .K. pneumoniae for subtyping using PFGE and RAPD methods. The results of that study showed 30 bands in the range of 15–7 bp and at the similarity level of 70%, 22 large clusters (W-A) at the similarity level of 85%, and 42 different groups were shown [K. pneumoniae to optimize subtyping using the PFGE method. In this method, the XbaI and AvrII enzymes were used. Both of these enzymes had a D-value greater than 99% for K. pneumoniae strains. The results of this study showed high repeatability and typography for both enzymes and indicated that electrophoresis parameters for each enzyme were proposed based on the size of limiting enzyme segments for the used equipment in order to obtain optimal results. The results of some studies are consistent with the results obtained in our study in some patterns [Ashayeri–Panah et al. investigated the genetic profile of 54 clinical isolates of re shown . Han et patterns .blaCTX-M-1 gene in 200 clinical isolates of K. pneumoniae. The PFGE and MLST differentiated the strains into 64 and 44 genotypes, respectively, indicating a high degree of differentiation of PFGE compared to the MLVA method [blaoxa-1 beta-lactamase-producing K. pneumoniae in a hospital in Spain. The relationship between K. pneumoniae strains was determined by PFGE and MLST. A distinct pattern of PFGE was observed with 5 different subtypes, and all isolates had a common pattern in the PFGE, which was characterized by the sequence of type 14 [Drakhshan et al. examined the genomic typing using PFGE and MLVA methods and examined the genetic diversity of the A method . Also, C type 14 .K. pneumoniae isolates recovered from outpatient and adolescent patients, PFGE analysis identified 18 clusters with clonal dependence in some cases [K. pneumoniae from serotype k1, which induces invasive abscess in the liver. More than 94% of the isolates were clustered in PFGE in a large group with a similarity of ≥70%. Within this group, 8 subgroups with ≥80% similarity were clustered. The findings of that study showed a very similar pattern in the PFGE method too which our study is in contrast with [In Salimizand et al.'s study that was carried out on 35 MDR me cases . Furtherast with .K. pneumoniae was investigated, in which PFGE differentiated the strains into 16 distinct genotypes and 5 clusters. In 20 isolates, the coefficient of similarity was more than 80%, and in 11 isolates, between each cluster, a high level of genetic heterogeneity was observed, which is consistent with our study [In a very recent study in Taiwan, over a period of 8 months, the molecular epidemiology of 31 isolates of ESBL-producing ur study .K. pneumoniae isolates is increasing. The presence of qnr (S and A) genes and point mutation of the gyrA gene were likely to be responsible for the resistance toward nalidixic acid and ciprofloxacin in our strains. Also, genotyping using PFGE indicated that the K. pneumoniae strains isolated in this study belonged to diverse clones.The results of this and similar studies indicated that the frequency of resistance to various antibiotics including quinolones in


The vital regulation of abnormally expressed lncRNAs in human cancers has been identified. This study is aimed at illustrating the role of LINC01426 in influencing malignant behaviors of lung adenocarcinoma (LUAD) and the possible mechanism. Differential expressions of LINC01426 in a downloaded profile containing LUAD and normal tissues were analyzed using Gene Expression Profiling Interactive Analysis (GEPIA) database and were reconfirmed in clinical samples collected in our hospital. In addition, LINC01426 level in lung carcinoma cell lines was detected by quantitative real-time polymerase chain reaction (qRT-PCR) as well. The relationship between LINC01426 expression and the age, tumor node metastasis (TNM) staging, lymphatic metastasis, tumor differentiation, and overall survival of LUAD was analyzed. After intervening LINC01426 level in H1299 and PC9 cells, proliferative and metastatic changes were assessed by functional experiments. LINC01426 was upregulated in LUAD tissues and cell lines. Its level was closely linked to TNM staging, lymphatic metastasis, tumor differentiation, and overall survival of LUAD. Knockdown of LINC01426 suppressed proliferative and metastatic abilities in H1299 and PC9 cells. LINC01246 is upregulated in LUAD samples and predicts a poor prognosis. It drives malignant process of LUAD via stimulating proliferative and metastatic abilities. Lung carcinoma is a globally prevalent malignant tumor . Non-smalncRNAs cannot be translated to proteins . They arIn the past decades, the involvement of lncRNAs in LUAD progression has been detected . For exain vitro and the possible mechanism.LINC01426 is previously identified to be upregulated in LUAD profiling . This sthttps://portal.GDC.Cancer.gov. Differential expressions of LINC01426 in LUAD tissues (n = 483) and normal tissues (n = 347) were analyzed using the R project.LUAD-TCGA profile was downloaded from LUAD and adjacent normal tissues were collected in Cancer Hospital and Shenzhen Hospital, Chinese Academy of Medical Sciences and pathologically confirmed. Tumor node metastasis (TNM) staging was defined based on the criteria released by the Union for International Cancer Control (UICC). Samples were frozen in liquid nitrogen and stored at -80°C. Included LUAD patients did not have chemotherapy or radiotherapy and signed informed consent. This study got approval by Ethics Committee of Cancer Hospital and Shenzhen Hospital, Chinese Academy of Medical Sciences, and experimental protocols conformed to the Helsinki Declaration.The human bronchial epithelioid cell line (HBE) and lung carcinoma cell lines  were purchased from Shanghai Institute of Biochemistry and Cell Biology . Cells were cultured in RPMI 1640 containing 10% FBS and 1% penicillin-streptomycin .Three lines of LINC01426 siRNAs  and negative control (si-NC) were purchased from RiboBio . Cells were grown to 60% of adherence and transfected using Lipofectamine 2000 .Qualified RNA was reversely transcribed into complementary deoxyribose nucleic acid (cDNA) using the PrimeScript RT reagent Kit . The cDNA was subjected to qRT-PCR using the SYBR Green Master Mix . Sequences were as follows: LINC01426: forward: 5′-TCCCGAAGTGCATAGGCAAG-3′, reverse: 5′-CCTGAACAAGTTGGGGAGGA-3′; GAPDH: forward: 5′-TATGATGATATCAAGAGGGTAGT-3′, reverse: 5′-TGTATCCAAACTCATTGTCATAC-3′.3 cells/well. At 0, 24, 48, 72, and 96 h, absorbance value at 450 nm of each sample was recorded using the CCK-8 kit  for plotting the viability curves.Cells were inoculated in a 96-well plate with 4 × 104 cells per well. 10 μl of EdU (50 μM) was applied in each well for cell labeling. Two hours later, cells were incubated with 4% methanol for 20 min, followed by PBS washing and incubation with Cell-Light™EdU Apollo®488 . 4′,6-Diamidino-2-phenylindole (DAPI) was used for nuclei staining in the dark. Finally, cells were washed in PBS and captured.Cells were inoculated in 96-well plates with 1 × 102 cells were inoculated in the 6 cm dish and cultivated for 14 days. Visible colonies were fixed in 4% paraformaldehyde for 30 min and stained using 0.5% crystal violet for 30 min. The number of visible colonies was counted.4 × 105 cells/ml and implanted in 6-well plates. Until 80% of cell attachment, an artificial wound was made using a sterilized pipette tip. Cells were washed in phosphate buffered saline (PBS) for 2-3 times and cultured in the medium containing 1% FBS. 24 hours later, wound closure was captured for calculating the percentage of wound healing.Cells were prepared into suspension with 1 × 103 cells were implanted on the top, while 600 μl of medium was applied on the bottom. After 48 h incubation, migratory cells on the bottom were reacted with 15 min methanol and 20 min crystal violet and captured using a microscope. Invasive cells were counted in 10 randomly selected fields per sample. Migratory cell number was similarly detected using Transwell chambers without Matrigel precoating.Transwell chambers  coated with Matrigel were used. 4 × 10t-test. Kaplan-Meier survival curves were plotted and compared based on LINC01426 level. P < 0.05 was considered as statistically significant.Data were expressed as mean ± standard deviation. Data processing was conducted by GraphPad Prism 6 . Each experiment was performed in triplicate. Differences between groups were compared using the Student's in vitro experiments, H1299 and PC9 cells were utilized.Through analyzing LUAD profiling downloaded from GEPIA database, it is found that LINC01426 was highly expressed in LUAD tissues than normal ones . ConsistClinical data of enrolled LUAD patients were recorded for analyses. No significant difference in LINC01426 level was found between LUAD patients over 60 years and whom younger than 60 years . Higher To explore biological functions of LINC01426 in LUAD cell behaviors, three lines of LINC01426 siRNAs were constructed. In particular, transfection efficacy of si-LINC01426-1 had the best efficacy, and it was used in the following experiments . KnockdoSubsequently, migratory ability in LUAD influenced by LINC01426 was assessed by wound healing and Transwell assay. After 24 h cell culture, weakened wound healing capacity was observed in H1299 and PC9 cells with LINC01426 knockdown than those of controls . In addiAccording to the latest report released by the International Agency for Research on Cancer, lung carcinoma remains one of the malignant tumors with the highest morbidity and mortality . Due to lncRNAs account for more than 98% of transcripts, and they are transcripts with limited or no protein-encoding functions . lncRNAsThere are still some obvious limitations in this study. For example, most of the analyses were performed based on the GEPIA dataset. The sample size of the patients collected from our institutions was small. What is more, lack of the in vivo experiments, such as tumorgenesis assay in nude mice, weakened the evidence level of our conclusions. In our future study, we will complete these assays to further explore the role of LINC01426 in the tumor growth of lung cancer.via stimulating proliferative and metastatic abilities.LINC01246 is upregulated in LUAD samples and predicts a poor prognosis. It drives malignant process of LUAD


Bifidobacterium breve CCFM1025 intervention significantly alleviated amyloid-β (Aβ)-induced cognitive impairment and inhibited neuroinflammation in mice. Moreover, we found that EE combined with B. breve CCFM1025 treatment restored AD-associated gut microbiota dysbiosis and reversed microbial metabolites changes. By integrating behavioral and neurological data with metabolomic profiles, we corroborated the microbiota–metabolite–brain interactions, with acetate and tryptophan metabolism as potential drivers. Taken together, our results provide a promising multidomain intervention strategy to prevent cognitive decline and delay the progression of AD through a combination of dietary microbiome-based approaches and lifestyle interventions.Alzheimer’s disease (AD) is characterized by behavioral and cognitive impairments and its increasing prevalence imposes a healthcare burden on society. To date, most intervention studies have only focused on a single AD-related factor and have yielded modest cognitive improvements. Here, we show that environmental enrichment (EE) training combined with  Bifidobacterium and Turicibacter, are decreased in abundance, whereas pathogenic taxa, such as Bilophila and Gemella, are significantly increased in patients with AD compared with healthy individuals (Akkermansia and Bifidobacterium (Alzheimer’s disease (AD) is a neurodegenerative disease characterized by the deposition of amyloid plaques, the formation of neurofibrillary tangles, cognitive impairment, and neuroinflammation . Accumulividuals . Furtheracterium . Indeed,acterium . Moreoveacterium . At the As determined by metabolomic screening, the gut microbiome generates various small molecule metabolites to communicate with the host . BroadlyLifestyle interventions to promote brain health, including diet, exercise, and social and intellectual stimulation strategies, can improve cognition in older individuals with AD . HoweverBifidobacterium breve CCFM1025 is a promising psychobiotic strain that has been isolated from fecal samples of healthy humans  L-cysteine. Freshly cultured bacterial cells were collected by centrifugation , and then washed and resuspended with 30% (v/v) glycerol. Aliquot resuspended bacteria cells in sterilized tubes and store at -80°C until use. The gradient dilution method was used to determine the number of bacteria cells. For oral administration, each aliquoted bacteria sample was washed twice and resuspended in phosphate-buffered saline (PBS) to reach a concentration of surviving bacteria at 1 × 109 colony-forming units/mL.Male adult C57BL/6J mice (8 weeks old) were purchased from the Model Animal Research Center of Nanjing University  and maintained at the Animal Center of Jiangnan University as previously described . The exp1-42) oligomer as previously described . The mice were administered 200 μL of bacterial suspension or sterile PBS as a vehicle control by gavage once a day for 6 weeks. The details of the treatment protocol for each group and the experimental timeline are shown in After acclimatization, the mice were divided into five groups. The AD model was established by intrahippocampal injection of an amyloid beta (Aβescribed . The vol3) and provided with running wheels, a red transparent mouse house, colored plastic tubes, wooden blocks, pressed cotton squares, and objects of different shapes , and interleukin 6 (IL-6) levels. For the analyses of synaptophysin (SYP), fibronectin type III domain-containing protein 5 (FNDC5), and transforming growth factor-β1 (TGF-β1), the supernatants were further diluted in cold PBS. All of these protein levels were measured using enzyme-linked immunosorbent assay kits from Elabscience  according to the manufacturer’s protocol.Hippocampal tissues were homogenized in pre-cooled PBS supplemented with protease inhibitor. The samples were centrifuged at 5,000 × g for 10 minutes at 4°C to collect supernatants. The supernatants were then used to measure AβBifidobacterium species was performed using primers designed to target the groEL gene. PCR products were purified and quantified using a Qubit dsDNA Assay Kit . Purified amplicons were pooled and paired-end sequencing was performed on a MiSeq PE300 platform .Microbial genomic DNA was extracted from fecal samples using a FastDNA Spin Kit . The V3-V4 region of bacterial 16S rRNA was then amplified using a universal primer pair (341F and 806R). The differential detection of via principal coordinate analysis (PCoA). The differential abundance of microbial taxa was calculated using linear discriminant analysis effect size (LEfSe) and visualized using a taxonomic cladogram tree. Metagenomes of the gut microbiome were computed from 16S rRNA sequences based on Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt), and functional pathways were predicted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology.Raw data were processed using the QIIME tools version 2.0 and bioinformatic analyses of the microbiota were performed. α-Diversity was calculated based on species richness and evenness using Chao 1, Shannon, and Simpson indices. β-Diversity was assessed based on the Bray–Curtis distance and was visualized For fecal metabolomic analyses, metabolites were extracted as detailed in Mars et al. . The met2SO4 (10% (v/v)) were added to each sample for acidification. Then, 800 μL of diethyl ether were added and mixed thoroughly. The supernatants were analyzed on gas chromatography–mass spectrometry  with a Rtx-Wax column .Fecal SCFAs, including acetate, propionate and butyrate, were extracted and quantified as previously described . For metP < 0.05 were considered to be statistically significant. Details of the statistical tests, the number of samples, and significance cut-offs are described in each figure legend. The network correlation between variables was determined using Spearman’s correlation coefficients and visualized with Cytoscape version 3.8.2 .Statistical analysis was performed using Prism version 8.0.2  and SPSS version 22 . Data are presented as mean values ± standard error of the mean or the median ± interquartile range. Before further statistical analyses, data were tested for normal distribution using the Shapiro–Wilk test and Q-Q plots. A one-way analysis of variance (ANOVA) with Holm–Sidak test or Student’s t-test was used for the parametric analysis of differences between groups and a Kruskal–Wallis test followed by Dunn’s test or Welch’s t-test was performed for nonparametric data variance analysis. Values of B. breve CCFM1025 combined with EE on AD-associated cognitive impairment, we performed behavioral tests in AD mice treated with or without either B. breve CCFM1025 or EE for 6 weeks after intersection hippocampal injection of Aβ1-42  were more prevalent in the EE + B. breve CCFM1025 group  and hierarchical cluster analysis indicated distinct clusters of metabolites in fecal samples  were identified in fecal samples. Aβ-injection induced significant differences in fecal metabolites, with model and control group clustering separately .The data presented in the study are deposited in the National Center for Biotechnology Information repository, accession number PRJNA847039 , the Fundamental Research Funds for the Central Universities .Thanks for the help of the Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


After propensity score-matching, there were 68 patients in each group. Among locally advanced PC patients, treatment with RP had a higher risk of biochemical recurrence compared to the RT group. In multivariate Cox regression analysis, treatment with RT plus ADT significantly decreased the risk of biochemical failure , but there was no significant difference in local recurrence, distant metastasis and overall survival . This information supported a clinical benefit in BCR control for patients undergoing RT plus long-term ADT compared to RP.To compare clinical outcomes between the use of robotic-assisted laparoscopic radical prostatectomy (RP) and radiotherapy (RT) with long-term androgen deprivation therapy (ADT) in locally advanced prostate cancer (PC), 315 patients with locally advanced PC  were considered for analysis retrospectively. Propensity score-matching at a 1:1 ratio was performed. The median follow-up period was 59.2 months (IQR 39.8–87.4). There were 117 (37.1%) patients in the RP group and 198 (62.9%) patients in the RT group. RT patients were older and had higher PSA at diagnosis, higher Gleason score grade group and more advanced T-stage (all  There were over 1.4 million new cases of PC and 375,304 related deaths around the world in 20202. Diagnostic estimates of loco-regional PC are over 90% in the United States3. In Taiwan, 58% of newly diagnosed cases of PC had loco-regional disease and, 11% had locally advanced disease between 2004 and 20124. The management of locally advanced PC remains a challenge for urologists.Prostate cancer (PC) is the second most frequent malignancy diagnosis made in men and the fifth leading cause of death worldwide6. PSA values are also influenced by patient age and prostate volume. In the last decade, magnetic resonance imaging (MRI) of the prostate has become an essential diagnostic tool for local staging. The Prostate Imaging Reporting and Data System (PI-RADS) version 2 was updated in 2015 and developed to promote global standardization in the interpretation and reporting of the prostate MRI examination. Many studies have investigated the accuracy of MRI in local staging7.Traditionally, the risk of PC is stratified by serum prostate-specific antigen (PSA), Gleason score (GS) of the prostate biopsy and digital rectal exam (DRE). However, DRE is a subjective test with potential inter-observer variability and GS has shown discrepancy between prostate biopsy and radical prostatectomy specimens8. Both treatments incorporate a multimodality approach in this high-risk population to improve oncologic outcomes at the expense of distinct potential complications.Robotic-assisted laparoscopic radical prostatectomy (RP) with pelvic lymph node dissection and radiotherapy (RT) combined with long-term androgen deprivation therapy (LTADT) are currently standard treatment options for locally advanced PC9. On the other hand, RT with LTADT does not require hospitalization and has a lower risk of urinary incontinence but does convey a higher rate of radiation cystitis, bowel/rectal problems and possible side effects of ADT10.RP has the advantage of more accurate disease staging with fewer bowel/rectal problems compared to RT, while anesthesia risk and associated higher risk of impotence and incontinence are still of concern10. However, RT dose and duration of ADT were heterogeneous. One ongoing prospective randomized study, SPCG-15 trial, comparing primary RP and RT plus ADT in locally advanced PC might provide valuable information in this specific population in the future11. To date, optimal management remains uncertain in locally advanced PC. In this study, we aimed to compare clinical outcomes in locally advanced PC between the use of RP and RT combined with LTADT.Standard treatment options for locally advanced PC include RP with pelvic lymph node dissection or RT combined with hormone therapy, but the optimal therapy is still controversial. Several retrospective series found an advantage for RP but only on the basis of a low level of evidence, while others favored RT because of superior outcomes when adding LTADTBetween January 1, 2008 and November 31, 2018, 533 PC patients with clinically T stage 3/4 defined by MRI were analyzed. There were 160 patients in the RP group and 373 patients in the RT group. Thirty-eight patients being operated on at other hospitals, 2 patients undergoing neoadjuvant ADT, and 6 patients with adjuvant RT after operation in the RP group were excluded. We restricted patients to those with RT and 1.5–3 years ADT on the basis of NCCN guideline recommendations. A total of 166 patients were thus excluded. Three patients who experienced biochemical recurrence (BCR) during ADT treatment were also excluded. Nine patients lost to follow-up, with 3 in the RP group and 6 in the RT group, were also excluded. Of these, 309 patients were included in our study: 111 in the RP group and 198 in the RT group.p values were calculated and a level of < 0.05 was considered statistically significant.A review was conducted of retrospectively obtained clinical data taken from the electronic medical records. Patient information was anonymized and de-identified prior to analysis. For each group, descriptive statistics were used to summarize the clinical presentation  and clinical T stage by MRI). Continuous variables were shown as median (range) and categorical variables as number (percentage). The Mann–Whitney U-test was performed to determine statistical significance for continuous variables between the groups while chi-square test or Fisher’s exact test was used for categorical variables. Propensity score (PS) matching analysis was performed to reduce the selection bias in this observational study, achieving a more comparison between the two groups. The PS was calculated using a logistic regression model and covariates entered into the PS matching model were as follow: age at diagnosis, biopsy GS grade group, iPSA and clinical T stage. PS matching was performed using a 1:1 matching method. The macro language made best matches first and next-best matches next. The PS matching sample was under SAS 8.2 Knowledge of logistic regression analysis. Of these, 136 patients were included in our study. BCR was defined as two consecutive times of PSA ≥ 0.2 ng/ml in the RP group and rising PSA of 2 ng/ml above the nadir (Phoenix criteria) in the RT group. Local recurrence was defined as lymphadenopathy or tumor recurrence in the pelvis by computed tomography (CT) or MRI. Metastasis was defined as distant metastasis in imaging . Kaplan–Meier analysis was performed to analyze BCR-free survival, local recurrence-free survival, metastasis-free survival and overall survival. To avoid immortal-time bias, the elapsed time for BCR-free survival analysis was calculated from the end of ADT treatment in the RT group and the operation date in the RP group to the date of BCR . In locaThe study was approved by the Institutional Review Board and Ethics Committee of National Taiwan University Hospital (IRB 201911084RINC) and all methods were performed in accordance with relevant guidelines and regulations. Informed consent from all subjects could be eliminated in this retrospective study according to Institutional Review Board and Ethics Committee of National Taiwan University Hospital (IRB 201911084RINC) regulation.p = 0.542). RT patients were older than RP patients (p < 0.001). A total of 171 patients (86.4%) in the RT group were older than 65 years, compared to 51 (45.9%) in the RP group. RT patients had higher iPSA levels (p < 0.001) and GS grade group (p < 0.001). There were 91 patients (46.0%) in the RT group with iPSA levels ≥ 20 ng/ml and 21 patients (18.9%) in the RP group. A total of 138 patients (69.6%) in the RT group had GS grade group ≥ 3, compared to 56 (48.6%) in the RP group. RT patients also had more advanced clinical T stage (p < 0.001). In the RT group, 90 patients (45.4%) had T stage ≥ T3b, and 7 patients (3.5%) had T stage 4. In the RP group, 19 patients (17.1%) had T stage ≥ T3b and no patient had T stage 4. In the RP group, 55 (49.6%) clinical T3 diseases defined by MRI were downgraded to pathologic T2.Patient characteristics are summarized in Table p = 0.071). There was no difference in age (p = 0.335), iPSA level (p = 0.932), GS grade group (p = 0.941) or T stage (p = 0.383) between the two groups.After propensity score (PS)-matched, there were 68 patients in both groups. Patient characteristics were well balanced and are summarized in Table p < 0.001, Fig. p < 0.001). There was a higher risk of BCR in patients with more advanced MRI T stage (p = 0.014). GS grade group could predict BCR only in univariate analysis (p = 0.019) but failed to predict BCR in multivariate analysis (p = 0.065). In Kaplan–Meier analysis, there was no significant difference in local recurrence-free survival , seminal vesicle invasion (SVI) or overall stage T3 PC7. The pooled data for ECE, SVI and overall stage T3 detection showed corresponding sensitivity and specificity of 0.57 (95% CI 0.49–0.64) and 0.91 (95% CI 0.88–0.93), 0.58 (95% CI 0.47–0.68) and 0.96 (95% CI 0.95–0.97) and 0.61 (95% CI 0.54–0.67) and 0.88 (95% CI 0.85–0.91), respectively. Because of microscopic involvement confirmed by resected prostate specimen regardless of size, high specificity and low sensitivity of MRI were found on meta-analysis. To make comprehensive decision-making regarding preservation of neurovascular bundles, high resolution of pre-operative images with good sensitivity is crucial. Draulans et al. revealed that the use of MRI images instead of DRE upstaged clinical T stage (33%) and European Association of Urology (EAU) risk grouping (31%)13. MRI showed a higher sensitivity than did DRE for detection of non-organ-confined PC  in terms of corresponding pathologic T stage, and furthermore, with incorporation of MRI instead of DRE staging alone, the surgical treatment strategy would be altered in 27% of patients. In other words, DRE or transrectal ultrasonography (TRUS) alone is not accurate enough for local staging (T stage), and MRI is still one of the best imaging tools for assessing ECE in clinical practice14.The reason to choose MRI as a reference of the clinical T stage is to better illustrate extracapsular invasion and for comparability between surgery and radiotherapy groups. Conventionally, DRE has been the fundamental method to detect PC and is used as a clinical T stage reference. However, DRE is a subjective test and could not detect anteriorly located tumors. That is why multiple risk stratification models, combining PSA, GS and DRE, have been developed to better evaluate the risk of PC. In addition, DRE often overestimates tumor staging and may not evaluate locally-advanced disease accurately15. Roach et al. noted that RT combined with ADT has lower cancer-specific mortality (CSM), distant metastasis and biochemical failure, without increasing the risk of fatal cardiac effect in locally advanced PC16. However, the use of RP has increased gradually and the proportion of patients treated with RP or RT has changed over time. In the Surveillance, Epidemiology and End Results (SEER) database (2004–2014), Marco et al. identified 5500 cT3N0-1M0 PC patients and noted that CSM was significantly lower after RP compared to RT17. Another Swedish observational study, including 34,515 patients, showed that RP has better survival than RT. Younger men and those with intermediate- or high-risk localized PC benefit more from surgery during 15 years of follow-up18. Consistently, a higher PSA and older age were noted in the RT group in the current cohort. In summary, the pathologic reports of prostatectomy, the dose of RT and duration of ADT treatment were not described in the majority of studies, and the discrepancies might influence the CSM18. One ongoing prospective randomized SPCG-15 trial with a similar trial setting as the current study might deliver valuable information regarding this specific population in the future11.Historically, men with locally advanced PC have been managed mostly with RT with ADT, while RP has been discouraged due to concerns about positive surgical margin, inadequate local control and side effects19. However, there was no difference in local recurrence, overall survival or cancer-specific survival. On the other hand, more adverse events were noted in the adjuvant group, where 56% experienced grade 3 adverse events versus 40% in the observational group (p = 0.016). Adjuvant RT caused more toxicity and could impair quality of life. Because of the above reasons, all patients receiving RP in our cohort were observed following prostatectomy, which enabled us to compare the effect of surgery alone without adjuvant RT confounding. In our cohort, patients in the RP group had a higher risk of BCR compared to the RT group. This might be explained by the lack of adjuvant therapy in the RP group and the dose of RT. Mitchell et al. found that 59% of patients suffered from BCR (defined as a PSA level > 0.4 ng/ml) after RP for cT3 disease but that only 12.9% of patients received adjuvant RT in their study20. Aligned with the Mitchell et al. study, the current study also showed that 45% of patients suffered from BCR in the RP group. When trying to compare the therapeutic effect between RP and RT, the additional peri-operative RT in the RP group led to some bias, so the current study only included patients with RP alone.Hackman et al. noted that adjuvant RT following prostatectomy prolonged biochemical recurrence-free survival compared with RP alone21. Stephen et al. also showed that 57% of patients initially classified as D’Amico high-risk PC have organ-confined disease at RP22. In addition, discrepancies in GS have frequently been found between biopsy and RP, such as up to 52.2% of GS over 8 tumors at biopsy had score downgrading at RP23. In the Mayo Clinic, 26% of PC patients with clinical T3 were downgraded to pathologic T220. Similarly, the cT3–4 stage has shown to be inaccurate in up to 33% of cases at RP24. In our cohort, 55 (49.6%) clinical T3 diseases defined by MRI were downgraded to pathologic T2 at RP. In addition, 50 out of 60 (83%) and 42 out of 51 (82%) patients before and after 2015 respectively were found to have clinical T3a disease on MRI, and there was no statistically different prevalence (p = 0.891). Among these patients, 33 patients (55%) diagnosed before 2015 were downgraded to pathologic T2, compared to 22 patients (43.1%) diagnosed after 2015; and despite a trend toward decreased discrepancy, there was no statistical difference (p = 0.076). Reviewing the accuracy of MRI imaging for local staging of PC, MRI images demonstrated moderate sensitivity for clinical T3 following prostatectomy 7. The current meta-analysis shows that MRI has high specificity but low sensitivity7. Conventionally, radiologists have focused on high-specificity reading to minimize unnecessary exclusion of men from curative treatment. In addition, MRI is limited for detection of focal ECE, which might increase its discrepancy25.Comparing to RT plus LTADT, one of the irreplaceable benefits of RP for patients in locally advanced PC is the ability to acquire accurate pathologic staging. Pathologic staging provided more reliable information to guide adjunctive therapies based on more precise data than bio-clinical variables including clinical T stage, biopsy GS or PSA. Indeed, 22 to 63% of PC initially defined as high risk have been found to have organ-confined disease following RP8. In our study, nearly all patients received RT with 7800 cGY in 39 fractions. Nevertheless, the most ideal total dose of RT is still under investigation and could influence oncologic outcomes. Local failure after RT is an independent factor of overall survival, CSM and metastasis-free survival in high-grade localized PC26. Few randomized control trials (RCTs) have revealed that dose escalation (range 7400–8000 cGy) has a significant impact on BCR, metastasis and CSM29. There are still inconsistent data on the effect of oncologic outcomes. However, the MRC RT01 RCT demonstrated that dose escalation (7400 vs 6400 cGy) showed an advantage in BCR, but the advantage did not translate into the improvement of overall survival30. Francolini et al. noted that dose-escalated pelvic radiotherapy and boost on positive lymph nodes were effective approaches to improve BCR31. Locally advanced PC or lymph node positive disease might benefit from dose-escalation. Some uncertainty still exists regarding different subpopulations. A retrospective analysis of the US National Cancer Database, including 42,481 patients receiving RT, showed that dose escalation is associated with improved overall survival in patients with intermediate- or high-risk PC, but not with low-risk PC32.Local disease control using different doses of RT in patients with PC is a critical issue. The total dose of 6000–7020 centigray (cGy) is currently recommended for localized advanced PC on the basis of the most updated guideline29. The 5-year rates of 2 or greater rectum and genitourinary tract toxic effects were 21 and 12% in high-dose arm and 15 and 7% with 7020 cGy. When dose escalation was applied, the rates of severe late side effects (> grade 3) were 2–3% for rectum and 2–5% for the genitourinary tract33. Consistently, 7 patients (3.5%) suffered from severe radiation cystitis and received blood clot evacuation in our RT group. In terms of treatment-related complications, 25 cases of complication were recorded in the RP group with the majority no more than Clavien–Dindo Grade III, while 8 major complications were recorded in the RT group. Among the 25 complications in the RP group, 12 were Clavien–Dindo Grade I, 8 were Grade II and 5 were Grade III . In the RT group, 196 patients (98.99%) received high-dose RT with 7800 cGY in 39 fractions. Among the 8 major complications, 1 patient died because of refractory radiation proctitis bleeding, while 7 patients suffered from severe radiation cystitis and needed blood clot evacuation. In addition, among the 8 major complications, 6 patients received volumetric modulated arc therapy (VMAT) and 2 patients received intensity-modulated radiation therapy (IMRT). Furthermore, IMRT, a major step with a wider therapeutic index, could be obtained from refined assessment of radiation-induced morbidity at an individual level34. In summary, although high-dose RT with LTADT showed better biochemical control, the mid-term survival outcomes were similar to those in treatment with RP in our cohort, and the relatively higher complications rate should not be underestimated. The optimal treatment strategies still need a large cohort to determine the risk–benefit.Dose escalation might be related to more toxic effects. Michalski et al. noted that dose escalation (7920 vs 7020 cGy) showed higher rates of toxic effects35. It could be challenging for investigators to recognize when immortal time bias influences the outcome of analyses. When treating BCR as the outcome, the PSA level would be influenced by ADT treatment and interfered with the evaluation of outcome if immortal time bias was not well considered. However, there were scarce data exploring this bias in previous studies, and investigations retrieved from a database showed heterogeneous ADT duration36. In our cohort, patients in the RT group received pre-defined and consistent duration of 1.5–3 years of ADT. If we were to extend the duration of ADT treatment, the RT group would have longer BCR-free survival, which would influence the outcome. As a result, the distinct feature of the current study is that we calculated BCR time from the end of ADT treatment to avoid immortal-time bias.To the best of our knowledge, this is the first study to compare RP alone, without adjuvant RT, with RT plus LTADT and take the immortal-time bias into consideration. RP alone allowed us the opportunity to observe the natural course of disease after prostatectomy. The potential for immortal time bias, also known as guarantee-time bias, exists whenever an analysis that is timed from enrollment is compared across groups defined by a classifying event occurring sometime during follow-up37. Fourth, there were no records of Charlson Comorbidity Index and ECOG in our study. These factors might have led to unavoidable selection bias, influencing the choice of treatment methods, and somehow impacting survival. Fifth, erectile function and continence were closely related to the outcome of satisfaction. In our cohort, we only explored the oncological outcomes, and not the functional outcomes. Sixth, whether the clinical benefit in BCR control remains when compared to patients receiving RP plus adjuvant RT is beyond the scope of the current study. Finally, we used MRI T stage because clinical T stage by DRE had inherent bias due to its subjective nature and potentially caused variability. However, there was a lack of central review of the MRI images. After analyzing the discrepancy rate of MRI reports between different time periods, it remained stable with no statistical difference. We therefore believed that the inter-observer variation in the current study was limited because all the radiologists in this high-volume tertiary referral medical center were well experienced.Nevertheless, there were some limitations in our study. First, the retrospective, short follow-up period and small sample size limited extensive analysis and we could only analyze mid-term overall survival within 5 years. There were only 10 deaths (3.2%) in our cohort and no cancer-related death event for analysis with enough statistical power. Second, patient distribution was unbalanced in the two groups. Patients in the RT group were older and had higher iPSA, higher GS and more advanced T stage compared to patients in the RP group. However, the effect could be minimal after adjusting by PS-matched and multivariate analysis. Third, the matched sample size was less than 50% and the incomplete matching might influence our results. However, decreasing the sample size from 1000 to 40 did not alter Type I error rate and led to relative biases below 10% unless the true confounders related only to the outcome are not included in the PS modelAmong patients with locally advanced PC, treatment with RP had a higher risk of BCR but no significant difference in the risk of local recurrence, metastasis, and overall survival compared to RT plus LTADT therapy. There is insightful information supporting a clinical benefit in BCR control for patients undergoing RT plus LTADT compared to RP. A further prospective and long follow-up period study for patients with advanced PC would be necessary.Supplementary Figure S1.


Terminalia arjuna (Roxb. ex DC.) Wight & Arnot of the Combretaceae family is one of the most frequently approved and utilized medicinal trees in the traditional medicinal system, which was used for the treatment of a variety of diseases, including cardiovascular disorders. The present study aims to identify phytochemicals from T. arjuna, that do not exhibit any toxicity and have significant cardioprotective activity using an in-silico technique. Four different cardiovascular proteins, namely human angiotensin receptor (PDB ID: 4YAY), P38 mitogen-activated protein kinase , 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-Co A) reductase (PDB ID: 1HW9), and human C-reactive protein (PDB ID: 1B09), were used as target proteins to identify potential inhibitors using a virtual screening of the phytochemicals in T. arjuna revealed casuarinin as a potential inhibitor of all selected target proteins with strong binding energy. Furthermore, MD simulations for a 100 ns time scale also revealed that most of the key protein contacts of all target proteins were retained throughout the simulation trajectories. Binding free energy calculations using the MM-GBSA approach also support a strong inhibitory effect of casuarinin on target proteins. Casuarinin’s effective binding to these proteins lays the groundwork for the development of broad-spectrum drugs as well as the understanding of the underlying mechanism against cardiovascular diseases through in vivo and clinical studies. Cardiovascular diseases (CVDs) are the leading cause of mortality worldwide.  CVDs are predicted to kill 18.6 million people per year, accounting for 33% of all global deaths. More than 75% of CVD deaths occur in low- and middle-income nations  showed the best glide docking XP score −8.5 kcal/mol binding energy value with best fit simulation study. Study conducted by Liu et al. [50 values of 73.06, 15.42, and 249.3 μM, respectively. All peptides inhibited the ACE activity via a non-competitive mode. The binding free energies of AVKVL, YLVR, and TLVGR for ACE were −3.46, −6.48, and −7.37 kcal mol−1, respectively. Docking of HMG-CoA (PDB ID: 3CCZ) with guajavarin, was found to be least binding energy  resulted in formation of four hydrogen bonds with the residues and amino acids SER 684 (3.2 Å), LYS692 (2.4 Å), ASP 690 (2.7 Å), and LYS 691 (3.1 Å) respectively [T. arjuna with phosphodiesterase 5A, sodium-potassium pump, and β-adrenergic receptor showed that casuarinin showed multiple inhibitions on phosphodiesterase 5A and sodium-potassium pump, whereas pelargonidin on phosphodiesterase 5A and β-adrenergic protein targets [Talapatra et al.  reportedmedicine . Khan [4medicine  studied u et al.  identifi targets . Recentlwww.rcsb.org, accessed on 10 November 2022)  . The rethttps://pubchem.ncbi.nlm.nih.gov/, accessed on 10 November 2022). These selected molecules were pre-processed and conformers were generated using Schrodinger Ligprep . Pre-processing of ligands included the following tasks: conversion of 2D structures to 3D format, addition of hydrogen atoms, generation of tautomers and ionization states, neutralization of charged groups, and finally geometry optimization of the molecule using OPLS 2005 force field [The selected phytochemicals and synthetic drugs were obtained from Pubchem (ce field  (Table 5T. arjuna with target proteins was performed using Glide (grid-based ligand docking) program incorporated in the Schrödinger molecular modeling package with extra precision (XP). Extra-precision (XP) docking and scoring is a more powerful and discriminating method that takes longer to perform than SP. XP is intended to be utilized on ligand postures that have been proven to be high-scoring utilizing SP docking. XP also implements a more complex scoring algorithm that is “harder” than the SP GlideScore, with higher criteria for ligand–receptor form complementarity. This filters out erroneous positives that SP allows through. Because XP can penalize ligands that do not match well to the particular receptor conformation employed, we propose docking to many receptor conformations whenever feasible. The best pose based on binding energies for each ligand–protein interaction was further analyzed in Discovery Studio (DS) visualizer . From the interaction profile, the ligands showing high binding energy were further considered for the molecular dynamic simulations.Molecular docking of selected phytochemicals of −9 tolerance limit), with a cut off distance of 9.0, was used to analyze protein structures every 1 ns. An average structure from the MD simulation that corresponded to the production phase was used to calculate the stability. Additionally, the histogram for torsional bonds, the radius of gyration (Rg), the root means square deviation (RMSD), and the root mean square fluctuation (RMSF) were used to examine structural alterations in relation to the dynamic role of the receptor–ligand complexes [In order to investigate the structural stability of the receptor–ligand complexes that were formed by molecular docking, the Desmond programme version 2.0  was utilized ,51,52,53omplexes ,58,59.MM is the difference of the minimized energies of the protein–ligand complex, while ∆GSolv is the difference between the GBSA solvation energy of the protein–ligand complexes and the sum of the solvation energies for the protein and ligand. ∆GSA indicates the surface area energies in the protein–ligand complexes and the difference in the surface area energies for the complexes [The binding free energies of protein–ligand complexes have been calculated using MM-GBSA and molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) ,61. As aomplexes .http://www.swissadme.ch/, accessed on 15 November 2022). Further, the toxicity of selected compounds was analyzed using the Protox-II tool to ascertain their risk of drugability [Lipinski’s rule  was considered the primary factor for screening of the molecules, and it was evaluated using the SWISS ADME web server (gability . Figure T. arjuna which shows strong binding against all the proteins can be used to develop broad spectrum cardioprotective drug with no adverse effects. However, further in vitro and in vivo studies are needed to validate these results.In the present study, we targeted various cardiovascular proteins through an in-silico method using phytochemicals with a rationale to block their interactions. This approach can help block the disease target proteins to prevent cardiovascular diseases. This study found prominent interactions of casuarinin with multiple protein targets and can be used as a promising compound to treat different cardiovascular diseases. MD simulations were depicted as the best binding stability with correlative motions. Moreover, the distribution of hydrogen bonds and the energy contribution of all simulated complexes of casuarinin with target proteins were calculated through binding free energy. Most of the studies have targeted one or two cardiovascular targets, but in our study, we have selected multiple proteins, and found that casuarinin from


PAH gene variants. After routine genetic analysis methods were applied, approximately 5% of PKU patients were still not diagnosed with a definite genotype.Phenylketonuria (PKU) is a common, autosomal recessive inborn error of metabolism caused by In this study, for the first time, we identified PKU patients with unknown genotypes via single-gene full-length sequencing.The detection rate of PKU genotype increased from 94.6 to 99.4%, an increase of approximately 5%. The variants c.1199 + 502A > T and 1065 + 241C > A were found at a high frequency in Chinese PKU patients.PAH.Our study suggest that single-gene full-length sequencing is a rapid, efficient and cost-effective tool to improve the genotype detection rate of PKU patients. Moreover, we provides additional case data to support pathogenicity of deep intronic variants in The online version contains supplementary material available at 10.1186/s40246-022-00397-w. PAH variant [Phenylketonuria (PKU) is the most common congenital disorder of amino acid metabolism, with a global prevalence of 1:10,000 newborns [ variant . When th variant  and Chin variant  for PKU, variant .PAH gene detection, PKU patients can be identified more quickly and effectively.Genetic analysis is an effective means for early etiological diagnosis, treatment guidance and prenatal diagnosis of PKU patients. Based on Chinese guidelines  gene diaPAH has been included in carrier screening by ACMG [The American College of Medical Genetics and Genomics (ACMG)  suggests by ACMG .PAH variants, including Sanger sequencing, next-generation sequencing (NGS) gene panels and multiplex ligation-dependent probe amplification (MLPA). Through the analysis of the PAH gene variant spectrum in different populations, 87–96% of PKU patients can be identified by sequencing all exons and flanking intron regions [PAH alleles [At present, routine molecular genetic analysis is commonly used in the clinical detection of  regions . Exonic  alleles , 12. App alleles .PAH, 17.9% occur in introns or untranslated regions [PAH single genes in PKU patients with unknown genotypes and with deletion/duplication of PAH. We examined the use of single-gene full-length sequencing for characterizing the molecular genetics of PKU patients and provide additional reference data for variant detection of deep introns of PAH.An analysis of the genetic landscape of PKU found that among pathogenic variants of PAH gene molecular genetic analysis through the gene panel (including PAH and DNAJC12) by NGS and MLPA.Based on clinical features and newborn screening, a total of 687 patients were diagnosed with PKU at the Third Affiliated Hospital of Zhengzhou University from January 2016 to December 2021. All of them underwent PAH gene molecular genetic analysis; (3) having been excluded from tetrahydrobiopterin deficiency.We reanalyzed the clinical phenotypes and molecular genetic results of these PKU families to screen out patients with unknown genotypes. The inclusion criteria were as follows: (1) exhibiting PKU phenotypes or increased blood Phe concentrations (≥ 360 μmol/L); (2) having only a single deleterious heterozygous variant or no variants identified after PAH detected by MLPA analysis for sequencing . An Illumina HiSeq X Ten Sequencer  with 150 bp paired-end sequencing mode was used for sequencing the genomic DNA. The mean sequencing coverage was > 1000 × , and > 99.0% of bases for every sample were sequenced to at least 20 × coverage.http://picard.sourceforge.net/). The variants of single nucleotide variants (SNVs) and insertion-deletion (InDel) were detected by GATK (https://software.broadinstitute.org/gatk/). Variant annotation and interpretation were conducted by ANNOVAR [http://gnomad.broadinstitute.org/), the 1000 Genome Project (http://browser.1000genomes.org), “HUABIAO” whole-exome public database (https://www.biosino.org/wepd/index) and dbSNP (http://www.ncbi.nlm.nih.gov/snp); (2) in silico prediction algorithms, such as SIFT (http://sift.jcvi.org), FATHMM (http://fathmm.biocompute.org.uk), MutationAssessor (http://mutationassessor.org), and CADD (http://cadd.gs.washington.edu); and (3) disease and phenotype databases, such as ClinVar (http://www.ncbi.nlm.nih.gov/clinvar), HGMD (http://www.hgmd.org), and BIOPKU (http://www.biopku.org/home/home.asp). The impact of variants on the splice site was predicted by using Alamut® Visual Plus v1.1.The sequencing reads were aligned to the human reference genome (hg19/GRCh37) using BWA, and PCR duplicates were removed by using Picard v1.57 ; (3) having no evidence of benign impact correlation in ClinVar and HGMD databases; (4) having an allele frequency no greater than 5% in the gnomAD database. We analyzed the variants screened manually and the deep intronic novel variants detected in patients in this study, including analyses of genome location and carrier population.A total of 1282 variants were included in the PAHvdb database , and we screened the SNVs in deep introns and untranslated regions by manual screening. The screening criteria were as follows: (1) located on introns, 5′UTR or 3′UTR that distance exons > 20 bp; (2) having reliable supporting reports in PubMed  to 99.4% (683/687), an increase of approximately 5%. However, there were still four patients with unclear genotypes. Although they have identified some variants of uncertain significance, the pathogenicity of these variants may need more evidence . This variant is not present in population databases (gnomAD no frequency). In silico analysis by RESCUE-ESE and ESEfinder, it predicts this variant is probably damaging to the protein structure. But these predictions have not been confirmed by functional studies. Therefore, it has been classified as a uncertain significance .PAH Expert Panel Specifications for interpretation of genetic variants, these deep intronic variants were classified as likely pathogenic or pathogenic.Combined with the phenotypic analysis, 68.4% of the PKU patients with c.1199 + 502A > T were associated with cPKU, while those with c.1065 + 241C > A preferred mPKU. The variant c.707-59C > G was identified in two patients with mPKU. According to ClinGen PAH gene deletions or duplicates in these samples. By comparison with the PAH reference sequence, we obtained the exons and introns contained in these regions. The results of analysis of the five samples were consistent with MLPA analysis. It shows that the single-gene full-length sequencing can analyze large-scale deletion/duplication within gene.We selected five patients in Table  and deteThrough the supplementary detection of single-gene full-length sequencing, 683 of 687 PKU patients were completely genotyped. The variant types of the fully genotyped patients are summarized in Table After our manual screening, there were seven nonbenign variants of deep introns in the PAHvdb database. Three of them were also identified in our study. In addition, in our study, we found a novel variant c.706 + 629A > C that had not been reported previously. Therefore, we analyzed a total of eight variants listed in the Additional file Among these variants, seven were detected in Asian populations, including Chinese and Iranian populations. In terms of the location of the variants, all variants were located in the catalytic domain Fig. .Fig. 2AnPKU is a type of genetic disease with involvement of definite pathogenic genes and is treatable. Early, rapid and accurate genetic-based diagnosis is very important for subsequent patient treatment, genetic counseling and prenatal diagnosis. The previous diagnosis strategy suggested using sequencing analysis to detect variants in exons and flanking intron regions combined with MLPA to detect deletions and duplications .PAH has been used to identify PKU patients.In this study, we analyzed the genetic test results of 687 PKU patients and found that 5.4% of the patients could not be accurately diagnosed based on the previous strategy. Interestingly, full-length sequencing improved the diagnosis rate to more than 99% and can lead to rapid and efficient genetic-based diagnosis. To the best of our knowledge, this is the first time that single full-length sequencing of Full-length sequencing analysis suggested that deep intronic variants might be an important potential pathogenic mechanism of PKU. We found that nearly 90% of patients with previously undefined genotypes carried deep intron variants. Three deep intronic variants c.1199 + 502A > T, c.1065 + 241C > A and c.707-59C > G detected in our study were also reported in other variant spectrum studies of Chinese PKU patients , 17. Our3+ ion binding sites involved in the binding of a cofactor [In the PAHvdb database, a total of 31 variants of deep introns are included. According to the previous criteria, we screened 8 nonbenign variants. Compared with other populations in the world, the frequency of deep intronic variants seems to be high in Asian, especially Chinese, PKU patients. We found that all of these deep intronic variants were distributed in the central domain, which includes Fecofactor . The moscofactor , 19.PAH could cause a splicing defect by a novel mechanism involving U1snRNP binding downstream of the 5' splice site, resulting in exon 11 skipping and the formation of incomplete protein. Dericquebourg et al. [F8. These variants lead to the creation of a de novo acceptor or donor splice site, and the formation of pseudoexon retention with different sequence lengths in the intron regions. For PKU patients, we speculate that the difference in phenotypic severity caused by deep intronic variants may be related to their location and impact on RNA splicing.Furthermore, we analyzed the genotypic-phenotypic correlation of these deep intronic variant carriers. Approximately 70% of patients with c.1199 + 502A > T had a severe cPKU phenotype. However, c.1065 + 241C > A and c.707-59C > G were associated with a lighter phenotype in PKU patients. Based on previous studies of genotype–phenotype correlation in PKU patients , 20, it g et al.  analyzedPAH acted to strengthen a cryptic branching point, and minigene expression showed that pseudoexon retention appeared in intron 11. The mechanism of pseudoexon inclusion caused by deep intronic variants may be due to activation of noncanonical splice sites, alterations to the splicing regulatory environment or loss of splice site competition [PAH deep intronic variants is required.An increasing amount of studies have confirmed that deep intronic variants contribute to different diseases by affecting mRNA processing –25. The petition , 29. In petition . TherefoPAH to amplify exons, deep introns and untranslated regions. And the time and cost of this single-gene full-length sequencing is similar to that of gene panel.We analyzed data from 683 completely genotyped PKU patients, and deep intronic variants were carried in 4.8% of patients. Such variants cannot be detected by routine genetic analysis methods. WGS has been proven to be a powerful tool to identify causative variants residing outside coding regions in a variety of diseases , 32. HowPAH. Therefore, this method of single-gene full-length sequencing not only has a lower detection cost than WGS but can also cover more regions of the targeted gene than routine genetic analysis methods. And using this method, we can find more deep intronic variants and micro-structure variants.Moreover, our results indicated that the high-depth sequencing data obtained by full-length sequencing could also be used to analyze large-scale deletions/duplications within Even with single-gene full-length sequencing, four PKU patients (0.6%) did not have a clear genetic diagnosis. At present, phenylalanine metabolism may also be related to epigenetic factors . Li et aOur study offers proof that single-gene full-length sequencing can achieve rapid, efficient and cost-effective genetic-based diagnosis in PKU patients. We believe this sequencing technique can be implemented in medical practice to help with the diagnosis, genetic counseling, carrier screening and prenatal diagnosis of PKU patients.Additional file 1.Table S1: Other variants information of patients with unclear genotypes; Figure S1: MLPA results of patient 38-42; Table S2: Summary of nonbenign PAH variants in deep introns.