Previous radical prostatectomy (RP) is considered a relative contraindication to the laparoscopic approach for inguinal hernia repair (LIHR). This study aimed to compare feasibility, safety and outcomes for patients undergoing totally extraperitoneal (TEP) LIHR who have previously undergone RP.This single surgeon, case–control study was performed using a prospective database of all patients undergoing TEP LIHR between 1995 and 2020. Patients who underwent previous RP were identified and compared to matched controls. Pre-operative, operative and post-operative data were analysed. The type of RP, open, laparoscopic or robotic, was identified and operative outcomes compared between the three groups.p < 0.0001) and bilateral  LIHR. The majority of cases were performed as day stay procedures. There was no difference in immediate recovery parameters including time to discharge, complication rates, return to normal function, return to driving or post-operative analgesia. At 3 months of follow-up there was no difference in hernia recurrence for unilateral  or bilateral  LIHR, nor chronic pain as measured by patient awareness or restriction of activity. No differences in operative and post-operative outcomes were identified between the three types of RP, other than difference in operative time (p = 0.0336).6532 LIHR cases were identified. 165 had previously undergone RP and 6367 had undergone primary LIHR without prior RP. The groups were matched for age, demographics and co-morbidities. All operations were commenced laparoscopically, three converted to open in the LIHR + RP group and none in the LIHR group. Median operative time in patients with previous RP was longer, for unilateral (40 min vs. 21 min, Previous RP should not be an absolute contraindication for TEP LIHR. Although previous RP adds complexity, in experienced hands TEP LIHR can be done safely, with outcomes equivalent to patients who have not previously undergone RP. Prostate cancer is one of the most commonly diagnosed cancers in men and for localised cancer, RP is the treatment of choice . RP can The primary aim of this study was to compare the outcome of patients undergoing TEP LIHR post RP to a matched control group undergoing LIHR without prior groin surgery. The secondary aim was to compare the outcome of patients relative to the RP approach.This retrospective case–control study analysed the outcome of LIHR using the TEP approach in the context of previous RP. LIHR post RP was compared to primary LIHR operations between 1995 and 2020, using information obtained from the prospective database of a single surgeon at a private surgical unit in Auckland, Aotearoa New Zealand. Institutional approval was obtained for the study.n = 165) included LIHR post RP (LIHR + RP). The control group (n = 6367) included primary LIHR operations only. Patients undergoing unilateral and bilateral LIHR were included. Bilateral repair was counted as two hernia repair operations and compared like for like in the analysis.A total of 6532 laparoscopic hernia repair operations were identified and assessed for eligibility Fig. . OperatiExclusion criteria: females, non-inguinal hernia repair operations , chronic groin pain without herniation (groin strain/sportsman’s hernia), trans-abdominal (TAPP) approach, patients undergoing a concurrent additional operation which might confer an increase in morbidity and, for the LIIHR group, age ranges outside of age < 50 or > 87 years. Exclusion of ages outside of this range aligned the LIHR group with the age range of the LIHR + RP group, reducing any potential confounding factors. Operations were excluded in the sequence detailed above to ensure an excluded operation was only counted once.In the LIHR + RP group, 13 operations were excluded . Exclusions were for the following: femoral hernia (1), groin strain/sportsman’s hernia 2), additional surgical procedure (8) and a TAPP approach (2). The remaining 152 LIHR + RP operations were included in the analysis, 128 unilateral and 24 bilateral (140 patients with prior RP). The types of initial RP techniques utilised were open (93 of 140), robotic (18 of 140) or laparoscopic (17 of 140) with 12 patients having unspecified type of prior RP repair . Exclusions were for the following: female patient (340), femoral hernia (66), groin strain/sportsman’s hernia (225), a TAPP approach (39), additional surgical procedure (244) and age < 50 or > 87 years (1729). This left 3724 LIHR operations included in the analysis 2234 unilateral and 1490 bilateral (2979 patients).Access to the extraperitoneal plane was gained through a short vertical incision in the ipsilateral anterior rectus sheath just below the level of the umbilicus, with introduction of a 0° 10 mm laparoscope. Two 5 mm operating ports were placed in the lower abdominal midline below the umbilicus, via the linea alba. Balloon dissection was not used to reduce the chance of peritoneal tearing against post-RP scarring. The extraperitoneal space was developed working from lateral, where tissues have not been previously dissected, to medial, where RP scarring is anticipated. A combination of sharp and blunt dissection was employed, taking care to dissect against the posterior wall of the inguinal canal in areas of scarring, in order to avoid inadvertent injury of any adherent bladder or bowel. Considerable care was exerted when dissecting close to the iliac vessels. A 15 × 10 cm polypropylene mesh was fixed to the periosteum of the superior pubic ramus and the linea alba using penetrative titanium tacs. Fixation laterally was not employed in order to allow any subsequent mesh contraction to occur without impediment. The aim was for day stay surgery. Patients were encouraged to return to full activity, without any restrictions regarding lifting/straining. Follow-up consisted of clinical review in 10–14 days and telehealth consultation at 3 months. The surgeon’s mobile phone number was provided for ease of contact if needed.Quantitative and qualitative pre-operative, operative and post-operative variables were assessed immediately prior to surgery, at operation, at clinical follow-up (10–14 days post-operatively) and by telehealth 3 months post-operatively. Pre-operative variables included age, sex and body mass index (BMI). Clinical variables assessed by the surgeon included hernia location (right or left), size , whether the hernia was direct or indirect, and if the patient had previously undergone hernia repair. Operative variables included the type of hernia, presence of lipoma of the spermatic cord, duration of operation (incision to wound closure), perceived difficulty of operation by surgeon  and details of any concurrently performed surgical procedure. Post-operative variables examined included hospital stay  and any post-operative complications or readmission. Patient perceived variables included the number of days until return to normal function, return to work, resumption of driving and the number of days simple analgesia was required . At three months patients had phone call follow-up and were asked if they had any residual awareness of the repair and if they had any restriction in movement, or function, as a result of their operation. Awareness and restriction were classified by the patient as nil, mild, moderate or severe. Any presence of hernia recurrence was also recorded. Clinical review was offered to all patients.T-tests for parametric numerical data and the Kruskal–Wallis and Dunn’s test for multiple comparisons . A p value < 0.05 was considered statistically significant.Descriptive statistics were used to characterise the groups, with mean and standard deviation (SD), or median and range. Groups were compared using Fisher’s Exact and Chi-squared test for categorical data, the Mann–Whitney test for non-parametric numerical data, unpaired p < 0.0001) between LIHR and LIHR + RP. The median age was 62 years for both unilateral (IQR = 14y) and bilateral (IQR = 11y) LIHR, compared to 68 for unilateral (IQR = 9.8y) and 71 for bilateral (IQR = 11.8y) LIHR + RP. There was no difference in BMI  or weight  between LIHR and LIHR + RP groups. There were differences in the rates of previous hernia repair with 17.1% of unilateral LIHR + RP operations identified as having a previous hernia repair on the contralateral side, compared to 10.5% for unilateral LIHR (p = 0.0079). There was no difference in rates of previous hernia recurrence .There was a difference in age  undergoing open RP, 13% (18 of 140) robotic and 12% (17 of 140) laparoscopic (Table p < 0.0001). The median operative time was 30 min (range 16–105) for bilateral LIHR and 71 min (range 31–111) for bilateral LIHR + RP, a 41-min difference (p < 0.0001). Operative time between open, robotic and laparoscopic unilateral LIHR + RP displayed a difference (p = 0.0336), with a median of 40 min (range 20–114) for open RP, 39.5 min (range 22–100) for robotic RP and 59 min (range 20–74) for laparoscopic RP. Comparison analysis by RP technique demonstrated a difference in operative time between laparoscopic vs. open (p = 0.0355) but no difference between laparoscopic vs. robotic (p = 0.0937), or open vs. robotic (p > 0.9999).Three of the first 13 cases in the LIHR + RP series required conversion to open. These have been excluded from results analysis when looking at outcomes of (completed) LIHR + RP. The median operative time was 21 min (range 9–120) for unilateral LIHR and 40 min (range 20–114) for unilateral LIHR + RP, a 19-min difference , side of herniation in unilateral cases (p = 0.7288) or element of lipoma  between LIHR and LIHR + RP. Indirect hernias were more common in the LIHR + RP group, 88% of unilateral and 68% of bilateral, compared to the LIHR group, 65% and 30%, respectively . The majority were discharged on the day of surgery, 98.6% of unilateral LIHR, 97.9% of bilateral LIHR, 94.5% of unilateral LIHR + RP and 91.7% of bilateral LIHR + RP. Only 10 patients (0.26%) were discharged at > 48 h, none LIHR + RP. Complication rates were not different between LIHR and LIHR + RP . Complications  occurred in 2.9% of unilateral LIHR, 1.6% of bilateral LIHR, 2.1% of unilateral LIHR + RP and 8.3% of bilateral LIHR + RP. No complications resulted in loss of life or serious morbidity. Return to normal function was not different between LIHR and LIHR + RP , with a mean of 3.5 days (range 0–27) for unilateral LIHR, 3.8 days (0–18) for bilateral LIHR, 3.5 days (0–10) for unilateral LIHR + RP and 5.1 days (0–14) for bilateral LIHR + RP. Return to driving was not different between LIHR and LIHR + RP , with a mean of 2.2 days (range 0–11) for unilateral LIHR, 2.3 days (0–10) for bilateral LIHR, 2.6 days (0–10) for unilateral LIHR + RP and 2 days (1–3) for bilateral LIHR + RP were reported. Analgesic use was not different between LIHR and LIHR + RP , with a mean of 2.2 days (range 0–21) for unilateral LIHR, 2.6 days (0–14) for bilateral LIHR, 2.7 days (0–16) for unilateral LIHR + RP and 3.2 days (0–10) for bilateral LIHR + RP.Time to discharge was recorded as day of surgery, the day following surgery or > 48 h post surgery, with no difference between LIHR and LIHR + RP , with 89.4% of unilateral LIHR, 90.8% of bilateral LIHR, 92.0% of unilateral LIHR + RP and 88.9% of bilateral LIHR + RP reporting no awareness. There was no difference in awareness by RP technique, with awareness reported in 7.3% of open, 12.5% of robotic and 6.7% of LIHR + RP (p = 0.7671). There was a difference in restriction between unilateral LIHR and LIHR + RP, with restriction reported in 0.06% of LIHR vs. 0% of LIHR + RP (p = 0.0006), but no difference between bilateral LIHR and LIHR + RP with no restriction reported in either group (p = 0.0563). There was no difference in restriction by RP technique, with no restriction reported after any of the LIHR + RP operations. Patients reported any awareness or restriction as mild, moderate or severe, with no patients reporting these as moderate or severe.Awareness and restriction were assessed categorically three months post-op, as no awareness versus awareness, and no restriction versus restriction, using the Chi-squared test. Losses to follow-up were 4.3% of unilateral LIHR, 10% of bilateral LIHR, 11.4% of unilateral LIHR + RP and 25% of bilateral LIHR + RP. There was no difference in awareness between LIHR and LIHR + RP . Recurrence was reported in 0.27% of unilateral LIHR (6 of 2234), 0.2% of bilateral LIHR (3 of 1490), 1.6% of unilateral LIHR + RP (2 of 128) and 0% of bilateral LIHR + RP cases (0 of 24). Of the two LIHR + RP recurrences, the first was considered a recurrence clinically, but on surgical exploration consisted of a small lipoma only. If this was excluded, unilateral LIHR + RP recurrence decreases to 0.78% (one in 128).There was no difference in hernia recurrence between LIHR and LIHR + RP (unilateral Previous RP, whether open, laparoscopic or robotic, should not be considered an absolute contraindication to TEP LIHR. In this series, no difference in morbidity, mortality or recovery was demonstrated when comparing LIHR and LIHR + RP, for either unilateral or bilateral repairs.These findings are supported by the limited literature available on TEP inguinal hernia repairs. One systematic review published in 2019  and two While publications on LIHR + RP are currently dominated by the TAPP method, there is no evidence supporting the superiority of the TAPP or TEP approach to hernia repair , 6, 9–13There is a significant learning curve to LIHR, whether TAPP or TEP is used. LIHR following RP confers an extra layer of potential difficulty due to scarring of the extraperitoneal plane over the posterior wall of the inguinal canal. This is reflected in the surgeon of this series needing to convert three of his first 13 LIHR + RP cases to open, despite having done nearly 400 LIHR before embarking on his first LIHR + RP case. He recommends that any decision to undertake LIHR post RP should only be made once a high level of competency with LIHR has been attained. There is no set number of cases to achieve competency. Surgical ego should not prevent sensible decision-making regarding conversion to, or selection of, open repair. The outcome of longer operative time for LIHR + RP in this series reflects the more challenging surgical environment post-RP secondary to RP scarring, even in experienced hands. While there is increased difficulty in performing LIHR + RP, these results support that this is a hernia repair that can be performed by the experienced laparoscopic surgeon –17.The dominance of indirect over direct inguinal hernias in the LIHR + RP group is explained by post-RP scarring, which is usually confined to the posterior wall of the inguinal canal, medial to the deep inguinal ring Fig. . This scAlthough the surgical literature considers RP a major risk factor for inguinal hernia formation, in this series 18.75% of unilateral LIHR + RP patients had a previous IHR on the contralateral side, compared to 10.47% of LIHR alone , despiteThe results of this series allowed for some comparison of the impact that the initial RP surgical technique—open, laparoscopic, or robotic—has on subsequent LIHR outcomes. Although the numbers in this study are limited, they suggest that initial RP technique does not impact on post-operative variables of awareness, restriction or recurrence in LIHR. The amount and density of scarring post RP varied from case to case, with no apparent influence by original RP technique. As in many parts of the world, robotic RP is becoming the dominant technique in Aotearoa New Zealand, but robotic RP did not appear to confer any increased ease of subsequent LIHR compared to open or laparoscopic, though the numbers in this series are very small for meaningful analysis. We acknowledge that these are findings that would need larger numbers and further research to form any definitive conclusions, but these results do seem promising that all patients who have undergone prior RP, regardless of RP type, should be considered for LIHR. To our knowledge, this is the first publication to look at the impact of initial RP type on subsequent hernia repair.While this study has several limitations, it also demonstrates many strengths. Its largest limitation is that it is based off observational retrospective data. It is also limited in that there was only three-month follow-up and it is plausible that some hernia recurrence, an important post-operative variable, was missed consequently. However, the variable being examined, prior RP, would make an RCT-based study impossible on ethical and practical grounds and all similar studies of this topic by nature will be observational retrospective studies. The large number of cases, 3876 overall, 152 LIHR + RP, gives this study strength and reduces the risk of bias despite its observational structure. Using data from a single surgeon, over a period of 25 years, removes intra-operative variability due to the surgical techniques and variances between surgeons.In summary, previous RP should not be considered an absolute contraindication for LIHR. Although previous RP adds complexity, in experienced hands TEP LIHR can be done safely, with outcomes being equivalent to patients who have not previously undergone RP.


SD 1.72) were enrolled from pediatric offices and educational settings into a randomized clinical trial . PCIT families demonstrated a significant reduction in child disruptive behaviors, increase in positive parent–child communication, improvement in child compliance, and reduction in parental stress compared to the control group. Exploratory analyses revealed no differential treatment response based on ASD severity, receptive language, and age. Results are promising for the use of PCIT with children demonstrating disruptive behaviors across the autism spectrum.A relatively large number of children with autism spectrum disorder (ASD) exhibit disruptive behavioral problems. While accumulating data have shown behavioral parent training programs to be efficacious in reducing disruptive behaviors for this population, there is a dearth of literature examining the impact of such programs across the range of ASD severity. To evaluate the effectiveness of Parent–Child Interaction Therapy (PCIT), an evidence-based treatment for children with problem behaviors and their families, in reducing disruptive behaviors among children (4–10 years) with ASD . Fifty-five children (85.5% male, 7.15 years;  Autism spectrum disorder (ASD), a continuum of neurodevelopmental disorders, is characterized by social deficits, communication impairments, and rigid, repetitive behaviors APA, . Over thThe presence of clinically significant behavioral problems among children with ASD is widely acknowledged and cited; however, the exact prevalence varies greatly due to the frequent use of clinical samples in studies , Early Start Denver Model, DIR/Floortime, Applied Behavioral Analysis (ABA), and ABA-derived models such as Early Intensive Behavioral Intervention (the UCLA Young Autism Project), Pivotal Response Treatment, and Positive Behavior Support  with ASD . This study expands the PCIT effectiveness research by including children with varying levels of ASD and who may fall outside the standard age-range for PCIT. The study included three hypotheses: (1) PCIT will result in a significant decrease in parent-reported disruptive behaviors; (2) parent and child interactions (child compliance rates and parenting skills) will significantly improve over the course of PCIT; and, (3) PCIT will result in significant improvements in parent efficacy and parent mental health (stress and depression). The second goal of the study was to perform exploratory analyses to assess the differential impact of the full PCIT protocol by autism severity, medication use, and language level on the disruptive behaviors of children across the autism spectrum. Finally, this study assessed parental satisfaction with PCIT.The study design followed a step-wise model for conducting psychosocial interventions for ASD, as outlined by Smith et al.  and in aN = 55 families) and their 4- to 10-year-old children participated in the present study. All adult caregivers living in the home were encouraged to participate in this treatment as research suggests that dual-parent involvement  leads to better maintenance of treatment gains . Their racial/ethnic composition was 65.5% White, 16.4% Black, 9.1% Latinx, 9.1% other ethnicity. Most children (80%) were referred by pediatric health care professionals, 12% were referred by teachers, and 8% were self-referred. At the time of intake, 56.4% were prescribed medication to address behavioral issues. Most children came from families with total household incomes between $50,000 and $99,000 . To be included in the study, children had to demonstrate at-risk or clinically significant externalizing behaviors . No parents endorsed concerns which was confirmed through a check of the children’s medical records. However, no cognitive data was collected in the present study.Children who participated in the study Table  were mosn = 8), (b) lacked severe behavior problems (n = 10), (c) were not previously diagnosed with ASD by a health professional (n = 4), or (d) the family did not complete the intake process (n = 9). There were three families screened as eligible for the study that cancelled their participation from the original group. Families not meeting the inclusion criteria (n = 37) were given feedback and appropriate recommendations for services. Upon completing the clinical intake, children were stratified based on the dichotomized variable of psychiatric medication use (yes/no), then matched using the continuous variables of externalizing behaviors (BASC), severity of autism (as measured using the CARS), and age. Families were assigned using stratified randomization to the control or treatment (PCIT) group using a research randomizer program. Randomization was determined by the lead researchers (first and second authors). Two children did not match and were not included in the randomization process, yielding a total of 30 treatment group (TG) families and 25 control group (CG) families. Treatment for children with ASD in the study location was limited. Many families were on a wait-list of 12–18 months for ABA. Families in both the TG and CG continued routine community care throughout the study, which primarily consisted of speech therapy services .Overall, 181 interested families responded to study recruitment. Please see Fig. The study was approved by the Eastern Virginia Medical School and the Children's Hospital of the King's Daughters Institutional Review Boards. Children were recruited by providing study information packets to pediatric offices, the Tidewater Autism Society of America, Community Service Boards, and school systems. Children were screened prior to enrollment on level of disruptive behavior (BASC), autism severity (CARS), and receptive language (PPVT). Enrolled families completed a baseline assessment (Time 1: pre-treatment) that included parental report questionnaires and parent–child observations which were videotaped and coded by research personnel. Approximately 8 weeks after the Time 1 assessment, all TG and CG families were scheduled to complete an interim-treatment assessment (Time 2). This interim assessment marked the transition from CDI to PDI for treatment families. The Time 3 assessment occurred post-treatment (upon graduation for TG), 8 weeks after Time 2. All families were compensated for study participation at Time 1 and Time 3 assessment visits. Data collection and treatment delivery was conducted in a medical center. Enrollment, assessments, and treatment delivery were conducted by the research team.The BASC-2  or “severe” (scores at or above a T-score of 50). None of the children in the sample had intellectual disabilities; therefore, the CARS-2 Standard Form was delivered for children 6 years of age and younger unless the child 7 or older had a notable communication impairment. The CARS-2 High Functioning was delivered to youth 7 years of age or older in the sample. Children’s CARS-2 T scores ranged from 32 to 65, with the mean score of 48.3 (SD 8.3).The CARS-2 . The Problem Scale includes “yes” or “no” responses and measures how problematic the child’s behavior is for the parent. The Intensity and Problem scales yield test–retest reliability coefficients of .80 and .85 across 12 weeks, respectively, and .75 across 10 months. The ECBI has been normed for children with ASD (ages 2–12 years) with cutoff scores on the Intensity (x = 169) and Problem Scales (x = 23) being significantly higher than neurotypical comparisons  DPICS training to ensure reliability. Coders were considered reliable after attaining a .75 kappa for each of the “Do” and “Don’t” skills on five consecutive observations. Throughout the study, two researchers blinded to group assignment independently observed the same individuals for 50% of all sessions and maintained a .85 inter-rater reliability. unless families terminated before treatment completion. In both phases of treatment , therapists actively coached parents toward understanding of the therapeutic interaction skills as assessed during a 5-min parent–child observation (DPICS) at the start of the session. On average, families achieved CDI skills competencies in 6.2 sessions and PDI in 5.9 sessions. Throughout treatment, parents were asked to practice the skills at home daily in 5–10 min sessions, initially focusing on CDI skills and then incorporating PDI skills at times when a command was necessary. The therapists included a licensed clinical psychologist and a supervised post-doctoral clinical psychology fellow, each of whom attended a 40-h PCIT training conducted by a PCIT Global Trainer. All therapy sessions were videotaped, and 50% of the session tapes from each family were randomly selected and checked independently by two coders for integrity using the PCIT treatment manual checklist. Accuracy was 95% with the treatment protocol. In addition, supervision from a PCIT Global Trainer was received regularly.Mean scores on parent-report questionnaires as well as behavioral observation counts and ratios derived from the DPICS were primarily analyzed using Repeated Measures MANOVAs. This is a suitable technique given the uniformity of the assessment schedule. Additionally, it is preferable to the alternative of multiple univariate tests as it detects patterns between multiple dependent variables which may not otherwise arise in univariate tests. Additional univariate comparisons are presented as follow-ups to the MANOVAs, as are graphical displays of observed effects.Among the 30 TG families and 25 CG families that were initially enrolled in study, 25 TG and 19 CG families completed all three assessment time points (Time 1–3) in the study see Fig. . Signifiλ  = 47.28, p < .001; Partial η2 = .48). Univariate tests reveal that children receiving PCIT demonstrated lower intensity of behavior problems at Time 2  = 8.21, p = .006) and significantly increased that difference  = 30.76, p < .001) at Time 3  = 5.77, p = .007; Partial η2 = .238), revealing that children in the TG demonstrated significantly fewer problems at Time 3  = 8.41, p = .006), but not at Time 2. Overall, each child in the treatment group scored in the non-clinical level of the ECBI on both the Problems and Intensity scale at the conclusion of the treatment.The two parent-report measures that were used to assess disruptive and oppositional behavior observed by the parent in the home included the ECBI Table ; Fig. 2 λ  = 11.79, p < .001; Partial η2 = .40). Univariate tests revealed that children in the TG demonstrated lower levels of Externalizing Problems at Time 3  = 6.61, p = .014) but not at Time 2.When measured with the BASC Externalizing Problems subscale Table , a similλ  = 49.81, p < .001; Partial η2 = .73). Univariate tests indicated no statistically significant differences between groups at baseline with TG parents showing significantly more “Do” behaviors at Time 2  = 49.07, p < .001) and maintaining this change  = 299.19, p < .001) through Time 3 , parents in the TG showed significant increases over the three assessment periods  = 21.33, p = .001; Partial η2 = .54). Univariate tests indicated no statistically significant differences between groups at Time 1, with TG parents showing significantly fewer “Don’t” behaviors at Time 2  = 36.68, p < .001), and maintaining this change  = 63.53, p < .001) through Time 3  showed a similar but inverse pattern over time. Parents in the TG showed significant decreases in “Don’t” behaviors over the three assessment periods  = 15.85, p < .001; Partial η2 = .47) with significant relative improvement occurring at Time 3  = 21.22, p < .001). A very similar pattern was observed during the CU activity with the Time X Compliance being significant  = 7.99, p = .001; Partial η2 = .30) and the major improvement being observed for Time 3  = 10.07, p = .003).Parents and children were observed for compliance/command ratio during the PLP and CU periods of the DPICS Table . Treatmeλ  = 4.67, p = .016; Partial η2 = .20). No significant difference were found between the TG and the CG on the PLOC-SF (p = .072) or BDI-II .All TG children were divided into ASD severity groups according to CARS-2 scores at or above a η2 = .08, p = .076).On the ECBI Intensity scale, no significant differences were found between children on psychiatric medications and those not on psychiatric medications, regardless of whether they were in the TG or CG. Medication X Group X Time was not significant  or age impacted change in child behavior (post-treatment ECBI Intensity—pre-treatment ECBI Intensity). The findings were not significant.Findings on the TAI show that TG parents found the techniques helpful in regards to disciplining their child (91%) and teaching their child new skills (93%). All parents of children in the TG felt their relationship with their child was better than before the program and that their child’s behavior problems and compliance with parental commands and requests had improved. Ninety-three percent of the parents reported satisfaction with the progress their child had made in regards to their general behavior and many (86%) felt that the program helped with other general personal and family problems. These findings suggest that families of children with a range of ASD presentations were highly satisfied with PCIT.This study further demonstrates the efficacy of PCIT among children with ASD and co-occurring behavior problems. The treatment was provided to children with ASD  and the results demonstrated similar behavioral changes to children without ASD who received PCIT. Specifically, based on both parent-report and observation data, children in the TG demonstrated a significant reduction in challenging behavior at the completion of CDI, and this reduction continued through the completion of PDI. Furthermore, from Time 1 to Time 3, TG families used significantly more relationship-building skills and obtained more compliance from their children when giving them commands, compared to the CG. This finding is similar to other PCIT literature conducted with children with ASD  and range of ASD severity, the TG responded to PCIT in similar ways to children without ASD  to lend insight in PCIT’s effectiveness of youth with ASD and diverse intellectual levels.Although this study did not measure non-disruptive behaviors of autism, such as self-stimulation, eye contact, language, and social engagement, positive changes in these behaviors were both observed by therapists and reported by parents and teachers. Next steps include assessing these behaviors in the context of other child characteristics including autism severity, language level, and cognitive functioning.As PCIT did not significantly decrease parent depression, future studies should explore ways to improve these measures of parent-functioning, possibly through the addition of a parent psycho-education/treatment module or referral for individual parental therapy. A larger sample size would allow for more thorough analyses of other possible correlates, such as age and gender.In families with typically-developing children, PCIT has been found to provide treatment effects lasting up to two years . In addition, the therapy can effectively prepare children for other intense and focused ASD therapies requiring cooperation and attention (Masse et al.,


Timely identification of patients at risk of worse clinical outcomes is vital in managing coronavirus disease 2019 (COVID‐19). The neutrophil‐to‐lymphocyte ratio (NLR) calculated from complete blood count can predict the degree of systemic inflammation and guide therapy accordingly. Hence, we did a study to investigate the role of NLR value on intensive care unit (ICU) admission in predicting clinical outcomes of critically ill COVID‐19 patients.We conducted a retrospective analysis of electronic health records of COVID‐19 patients admitted to ICUs at Hazm Mebaireek General Hospital, Qatar, from March 7, 2020 to July 18, 2020. Patients with an NLR equal to or higher than the cut‐off value derived from the receiver operating characteristic curve were compared to those with an NLR value below the cut‐off. The primary outcome studied was all‐cause ICU mortality. The secondary outcomes evaluated were the requirement of mechanical ventilation and ICU length of stay (LOS).p < 0.001), chronic kidney disease (p = 0.03), malignancy (p < 0.002), and NLR ≥ 6.55 (p < 0.003). There was also a significant association between the requirement of mechanical ventilation  and increased ICU LOS  in patients with ICU admission NLR ≥ 6.55.Five hundred and nineteen patients were admitted to ICUs with severe COVID‐19 infection during the study period. Overall, ICU mortality in the study population was 14.6% (76/519). NLR on ICU admission of ≥6.55 was obtained using Youden's index to predict ICU mortality, with a sensitivity of 81% and specificity of 41%. Mortality was significantly higher in patients with age ≥60 years (Higher NLR values on ICU admission are associated with worse clinical outcomes in critically ill COVID‐19 patients. Despite being used for many years, new implications of CBC are still being explored, with neutrophil‐to‐lymphocyte ratio (NLR) being one of them. NLR is calculated from the white cell differential count as a ratio of the number of neutrophils divided by the number of lymphocytes. It is an inflammatory biomarker used in the early detection of sepsis,Coronavirus disease (COVID‐19) is an infectious respiratory system illness caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Initial cases were identified in Wuhan city, China, in December 2019. Since then, the infection quickly spread and was declared a pandemic by World Health Organization (WHO) on March 11, 2020.There is growing evidence that a hyperinflammatory response to SARS‐CoV‐2 results in rapid deterioration and worse outcomes in critically ill COVID‐19 patients.22.1We retrospectively analyzed electronic health record data of patients admitted to intensive care units (ICUs) at Hazm Mebaireek General Hospital, Qatar, between March 7, 2020 and July 18, 2020. Medical Research Center (MRC) at Hamad Medical Corporation, Qatar, validated the study protocol and waived the need for informed consent (protocol no.: MRC‐01‐20‐1155). The inclusion criteria were: COVID‐19 positivity established by reverse transcription polymerase chain reaction of nasopharyngeal swabs, age more than 18 years, and ICU admission for more than 24h.2.2Data collected were demographic characteristics, comorbid conditions, blood test results, need for mechanical ventilation, ICU length of stay (LOS), and mortality. The NLR for each patient was calculated by dividing the absolute neutrophil count (ANC) by the absolute lymphocyte count (ALC) obtained from the CBC test. The normal adult range of ANC was 2000–7000 per microliter, and for ALC, it was 1000–3000 per microliter. The primary outcome investigated was all‐cause ICU mortality in relation to NLR. The secondary outcomes evaluated were the requirement for mechanical ventilation and ICU LOS.2.3χ2 test and Fisher's exact test were used to compare categorical data. As the numerical data followed a non‐normal distribution, two‐tailed nonparametric tests, namely, Spearman's correlation and Mann–Whitney U‐test, were used for correlation analysis and comparison of mean ranks, respectively. The area under receiver‐operating characteristic curve was calculated to identify prognostic cut‐off points for mortality. Based on the best‐identified cut‐off point, NLR was dichotomized into two categories. Outcome predictors were obtained by multiple logistic regression (LR), which also controls for possible confounding bias. Alpha was set at 0.05 to determine statistical significance.The IBM Statistical Package for Social Sciences (SPSS version 24.0) was used to analyze the data. Numerical variables were summarized using mean ± SD or median (IQR) according to distribution, while categorical variables were summarized as frequencies or percentages. The 33.1During the study period, 519 patients with COVID‐19 infection were admitted to ICUs at Hazm Mebaireek General Hospital. The mean age of patients was 51.6 ± 13.6 (mean ± SD) years, and 93.3% (484/519) of them were males. The most frequent comorbid conditions were diabetes mellitus  and hypertension . Table 3.2p < 0.001), in patients with pre‐existing hypertension (p < 0.001), cardiovascular disease (p = 0.04), chronic kidney disease (p < 0.001), chronic liver disease (p = 0.03), malignancy (p < 0.001), and NLR ≥ 6.55 on ICU admission. Variables with p ≤ 0.25 were entered into the LR model using the forward LR method to eliminate confounders and insignificant factors . NLR cut‐off value predicting mortality, obtained using Youden's index, was 6.55 for NLR on ICU admission, with a sensitivity of 81% and specificity of 41%  of the study population required intubation, and the need for mechanical ventilation was significantly higher in patients with NLR ≥ 6.55 on ICU admission, as shown in Table 9 of the Rs) = 0.157 (p < 0.001). Similarly, there was a weak positive correlation between NLR on hospital admission and hospital LOS with Rs = 0.092 (p = 0.03).NLR median values on hospital and ICU admission were significantly higher among patients who died or required mechanical ventilation Table . A stati4Our single‐center retrospective study revealed a significant association of NLR value on ICU admission with ICU mortality, need for mechanical ventilation, and ICU LOS, with higher NLR values predicting worse outcomes.r = 0.738, p < 0.001) in COVID‐19 cases.NLR reflects the balance between innate (neutrophil granulocytes) and adaptive (lymphocytes) immune responses. The rise in neutrophil count and decline in lymphocyte count is a multifactorial dynamic process depending on finetuning and regulation of various immunologic, neuroendocrine, humoral, and biologic processes such as margination/demargination, mobilization/redistribution, accelerated/delayed apoptosis, the influence of stress hormones, and sympathetic/parasympathetic imbalances.p < 0.02), longer mean ICU stay  and more days utilizing mechanical ventilation .The physiological values of NLR in a healthy adult population depend on ethnicity and other factors like smoking, obesity, and diabetes mellitus. The average value of NLR observed in Afro‐American, Hispanic, and Caucasian individuals was 1.76, 2.08, and 2.24, respectively.Our study has some limitations. First, we exclusively studied NLR values in critically ill COVID‐19 cases, which is a tiny proportion of the total number of infected cases. Hence, our sample size was small, and also, we could not evaluate the relationship between NLR values and noncritical COVID‐19 patients. Second, due to the retrospective study design, we could not exclude the impact of treatments received before ICU admission on the outcome, which may have resulted in a potential overestimation of the NLR performance . Third, we did not have pre‐existing data on the average NLR values in the healthy local population compared with COVID‐19 cases. Finally, although the study was performed in a tertiary care COVID facility, it is a single‐center study among the predominantly male population (93.3%), thereby limiting its generalizability.5This retrospective single‐center study of 519 critically ill COVID‐19 patients revealed that the NLR value ≥6.55 on ICU admission was associated with higher ICU mortality and the need for mechanical ventilation. Despite the limitations, our results are in line with the previously demonstrated predictive ability of NLR and conducted in a previously unstudied patient population. Therefore, we believe that the trends and predictors shown here are likely real while realizing that external validation would strengthen our conclusions.Husain S. Ali: Conceptualization; methodology; writing – original draft; writing – review and editing. Dore C. Ananthegowda: Conceptualization; methodology. Ebrahim M. A. Ebrahim: Formal analysis; investigation. Nevin Kannappilly: Data curation; investigation. Mohammad Al Wraidat: Data curation; investigation. Ahmed S. Mohamed: Conceptualization; project administration. Mohamad Y. Khatib: Supervision; writing – review and editing.The authors declare no conflict of interest.6The study was approved by the MRC at Hamad Medical Corporation (protocol no.: MRC‐01‐20‐1155). The research was conducted per the ethical standards noted in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. No consent was obtained due to the retrospective nature of the study.The lead author Husain Shabbir Ali affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned  have been explained.


A large spontaneous splenorenal shunt (SRS) will greatly impact portal inflow to the graft during liver transplantation (LT). Direct ligation of a large SRS is an uncommon surgical procedure and the hemodynamic consequences of this procedure are unknown.In this retrospective study, we described our technique for direct ligation of a large SRS and the consequent hemodynamic changes during LT. 3-Dimensional computed tomography and Doppler ultrasonography were used to evaluate SRS and portal vein blood flow volume (PFV).p = 0.0009). In all ligation patients, the SRS was easily identified and isolated, it was located just below the distal pancreas and beside the inferior mesenteric vein. PV flow increased significantly from 68.74 ± 8.77 to 116.80 ± 16.50 ml/min/100 g (p < 0.0001) after ligation; this was followed by a reduction in peak systolic velocity of the hepatic artery from 58.17 ± 14.87 to 46.67 ± 13.28 cm/s (p = 0.0013).A total of 22 recipients had large SRS including 13 with PFV <85 ml/min/100 g (ligation group) and 9 with PFV ≥85 ml/min/100 g (no ligation group). The diameter of SRS was significantly larger in the ligation group than in the non-ligation group (22.92 ± 4.18 vs. 16.24 ± 3.60 mm; Direct ligation of large SRS was an effective and safe surgical procedure to overcome the problem of portal hypoperfusion during LT. Whole liver transplantation (LT) requires a hepatopetal portal inflow of at least 1000 ml/min to ensure proper liver function . The porSurgical procedures used for SRS during LT include renoportal anastomosis (RPA) , 10, 11,Three-dimensional computed tomography (3D CT) and Doppler ultrasonography (US) were used to evaluate the SRS. A large SRS was defined as a shunt with diameter >10 mm before transplantation. Between January 1, 2017 and December 31, 2020, 720 adults (age ≥18 years) underwent deceased donor LT at the Second Xiangya Hospital of Central South University. A total of 22 recipients (3.05%) who were found to have a large SRS  were retrospectively analyzed. The study protocol conformed to the ethics guidelines of the 1975 Declaration of Helsinki and was approved by Institutional Review Board of the Second Xiangya Hospital of Central South University (No. 2019–050). Written informed consent was obtained from all participants. The transplantations were performed according to the Declaration of Istanbul, and no executed prisoners were used as donors.Intraoperative and postoperative PV blood flow volume (PFV) was evaluated by color Doppler ultrasound (US). 3D CT was routinely performed before and after LT to assess portosystemic shunts. The Doppler US parameters for blood flow features were calculated using the Logiq P5 US System . PFV was calculated as cross-sectional area × portal blood velocity (angle-corrected) . Donor lThe operations were performed by the same surgical team with the same classical orthotopic technique or piggyback methods. The flowchart for the surgical management of large SRS is shown in Liver biopsy routinely performed at the end of liver graft harvesting and LT. The same (blinded) pathologist evaluated all liver samples. Grafts were also evaluated for the presence of ischemia reperfusion injurys (IRI). IRI defined by the combination of apoptotic hepatocytes and inflammatory neutrophilic polynuclear's infiltrate throughout the lobule and around centrilobular veins. An IRI was graded as absent, mild, moderate, or severe. Severe IRI required centrocentrilobular necrosis . 3D CT wp < 0.05 were considered statistically significant.Values were expressed as mean ± SD. Statistical analysis of hemodynamic consequences were performed with the Student's t test. SPSS 11.0  statistical software were used for statistical analyses and differences with The preoperative characteristics and flow measurements of cases with large SRS are shown in There were 13 patients with PFV <85 ml/min/100 g (ligation group) and 9 with PFV ≥85 ml/min/100 g (non-ligation group). As shown in p < 0.0001) after ligation (p = 0.0013), with no significant difference in PSV immediately after ligation as compared to day 7 post transplantation (p = 0.3173) in the non-ligation group.PFV was elevated in all cases with SRS occlusion and increased significantly from 68.74 ± 8.77 to 116.80 ± 16.50 ml/min/100 g ; at the time of writing, no HA flow-related biliary complications or SRS ligation-related kidney injury occurred in 13 patients, 21 of the 22 patients were alive with no evidence of PV complications. One patient (ligation group) died of severe infection during the perioperative period but this was unrelated to the procedure of SRS ligation.Spontaneous portosystemic shunts such as SRS are portocaval communications in patients with chronic liver disease and portal hypertension . A largeIn the present study, no SRS ligation-related kidney injury or HA flow-related biliary complications were occurred in 13 patients. For patients without PVT or with resectable PVT, LRVL is the recommended surgical treatment for SRS in LT, however, previous studies have shown that ligation of the left RV can lead to sustained and elevated serum creatinine levels and decreased kidney size in recipients , 18. RPAThe hepatic arterial buffer response is an intrinsic mechanism of the liver to maintain total hepatic blood flow when portal perfusion decreases . AdenosiIn all of our patients, the SRS was located below the distal pancreas and beside the inferior mesenteric vein, it was easily identified by locating and following the LRV. The retroperitoneal tissue was relatively loose, and the SRS was always free without extra branches, thus, the large SRS was not technically difficult to identify, nor was it dangerous to isolate. In the present study, no procedure-related complications occurred in patients who underwent direct SRS ligation. However, a patent PV is mandatory for direct ligation of the SRS. Additionally, SRS ligation should not be attempted in patients with unresectable PVT or in whom angioplasty of PV stenosis has failed.There were several limitations to the study. Firstly, it was designed as a retrospective analysis of a relatively small number of patients. Secondly, the judgment of portal vein blood flow by Doppler US is not very precise, while measuring PFV by ultrasonic flowmetry is more objective, this was not always possible at our center. Thirdly, the PFV reference value of 85 ml/min/100 g of our center was calculated from a historical non-SRS cohort of 100 deceased adult liver transplant donors. However, a previous study used PFV <1200 ml/min after reconstruction as a reference value to determine whether the SRS was disconnected by LRVL . ProspecIn conclusion, we demonstrated that direct ligation of a large SRS was effective in achieving nearly 2 folds portal inflow. The large SRS was easily identified and isolated, and no SRS ligation-related complications injury were found in the ligation group. Additionally, the surgical procedure for SRS ligation is in line with normal physiology and minimally invasive. Direct ligation of large SRS could be an effective and safe surgical procedure to overcome the problem of portal hypoperfusion during LT.


Leiurus macroctenus, on tissue proteolytic activity and MMP levels have not yet been conducted.  Scorpion stings may be life-threatening since their venoms are comprised of a wide range of toxins and other bioactive molecules, such as enzymes. At the same time, scorpion envenomation may increase matrix metalloproteases (MMPs) levels, which enhance proteolytic tissue destruction by venom. However, investigations on the impact of many scorpions' venoms, such as those of Leiurus macroctenus envenomation and evaluate the metalloproteases and serine proteases' contributions to the total proteolytic activity. Changes in MMPs and TIMP-1 levels were tested as well. Envenomation led to a significant increase in proteolytic activity levels in all assessed organs, mostly in the heart (by 3.34 times) and lungs (by 2.25 times).  The present study aimed to examine the total proteolysis levels in various organs after Leiurus macroctenus envenomation causes systemic envenomation, which may induce multiple organ abnormalities, mostly because of the uncontrolled metalloprotease activity. Since EDTA presence showed a noticeable decrease in total proteolytic activity level, metalloproteases appeared to play a prominent role in total proteolytic activity. At the same time, MMPs and TIMP-1 levels were increased in all assessed organs, suggesting that  Scorpions can be considered one of the most dangerous animals around the globe. Nearly 1.5 million envenoming cases are being reported annually, and this number may increase due to the rapid urban expansion .Leiurus abdullahbayrami venom's dry weight [Leiurus quinquestriatus venom also indicate the presence of various metalloproteases [Scorpion-derived venom components have been identified and described . Proteiny weight . The majy weight , 3, 5. Hroteases .Once inside the body, venom components trigger the inflammatory response, which involves the liberation of various proinflammatory and anti-inflammatory cytokines and the attraction and activation of immune cells , 8. VenoLeiurus  are extremely dangerous [Leiurus for a long time was considered monotypic, Lowe et al. have recently described a few new species, including Leiurus macroctenus [Scorpions of the genus angerous , but theangerous –19. Althroctenus , which croctenus .Leiurus macroctenus venom on proteolytic activity in tissues during envenomation or information about changes in tissue MMP levels after a Leiurus macroctenus sting.To the best of our knowledge, there is no data about the impact of particular enzymes present in Leiurus macroctenus venom injection and evaluate serine proteases and metalloproteases' contributions to the total proteolytic activity. Furthermore, we tested the MMPs' content level changes as a response to envenomation.The aim of this study was to examine the proteolysis levels in various organs after Leiurus macroctenus specimens were used. All scorpions were previously identified by Mark Stockmann and kept in private collection in Ibbenbüren (Germany). Scorpions were maintained separately in transparent plastic boxes (10 × 5 × 5 cm) layered with sand (Exo Terra « Desert Sand») by 1 cm. In the center of each box were placed water bowls with distilled water, which were refilled every week. All animal containers were kept in constant conditions . Proper aeration was reached by numerous holes in container. Specimens were fed upon 1 Shelfordella lateralis cockroach once a week, in case of refusal of food, cockroach was taken away in 2 days after feeding. Once a month, containers were cleaned of cockroach remnants.In this study, 10 mature The procedure of venom collection was performed according to Ozkan and Filazi's method , modifieμg/mL) (LD50 = 0.08 mg/kg), dissolved in saline solution (0.9%). The control group (13 rats) was injected with 0.5 ml saline solution (0.9%) alone. Organ isolation and homogenization were performed at 4°С, right after animal execution. Homogenization was conducted using 50 mM Tris-HCl (pH 7.4) buffer with 140 mM NaCl and 1 mM EDTA addition. The volume of buffer used was five times higher (in grams) than the mass of isolated organs. The crude homogenate was centrifuged at 600 g for 15 min, with further supernatant collection and its centrifugation at 15000 g for 15 min. Obtained homogenate aliquots were frozen in liquid nitrogen.The group, consisting of 60 albino male rats (180 g ± 3 g), were injected intramuscularly with 0.5 mL venom solution (28.8 pH 7.4) and 1.6 ml 1 M NaOH. The obtained mixture was left at room temperature for 40 min, and after that it was boiled in a hot water bath. The casein solution was cooled afterwards, pH 7.4 was adjusted by 1 M NaOH, and the total volume of 100 ml was adjusted by phosphate buffer. About 500 μL of homogenate was diluted with 0.05 M phosphate buffer (pH 7.4) to a total volume of 1 mL. After that, 1 mL of 4% casein solution was added, and the obtained mixture was incubated at 37°C for 30 min. The reaction was stopped by a 3 mL addition of 15% TCA, after which samples were centrifuged at 1000 g for 30 min. The supernatant was collected and used for absorbance measurement at 280 nm. The control sample mixture consisted of casein, phosphate buffer, and TCA in identical proportions. In order to evaluate metalloproteases and serine proteases' activities, 0.2 M ethylenediaminetetraacetic acid (EDTA) and 0.2 M phenylmethylsulfonyl fluoride (PMSF), respectively were added to the reaction mixture. Total proteolytic activity was expressed as rel. units·(mg of protein)−1.Total proteolytic activity was analyzed using the caseinolytic activity examination proposed by Kunitz  with somμg/mL with 0.05 М Tris-HCl buffer (pH 7.4) and incubated overnight at 4°С in sterile ELISA plates wells. After incubation, plates were washed with immobilization buffer and blocked for 1 hour at 37°С by 5% nonfat dry milk solution to avoid nonspecific binding. After that, plates were washed again with buffer containing 0.1% Tween-20, with further incubation with the corresponding primary antibodies  (1 : 3000) for 1 hour at 37°С. Next, plates were washed with buffer containing 0.1% Tween-20 and incubated with HRP-conjugated secondary antibodies  (1 : 6000) for 1 hour at 37°С. After that, wells were washed with 0.1% Tween-20 buffer and incubated with 0.4 mg/ml o-phenylenediamine, diluted in 0.05 M citrate-phosphate buffer, in the presence of 30% Н2О2 to visualize the reaction. The reaction was stopped 10 minutes later by adding 100 μL 1 М H2SO4. The optical density was measured at 492 nm using a microplate reader.Matrix metalloproteases quantification was conducted using immunoenzyme analysis standard method for soluble proteins . The samProtein contents were measured by Bradford assay .∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001, differences between groups were considered statistically significant.The values present in tables and figures are expressed as mean ± SEM. The significance of differences between the means of experimental groups was determined by one-way analysis of variances (ANOVA) with Tukey's multiple comparisons test, performed in GraphPad Prism 9. When Leiurus macroctenus venom injection and to evaluate metalloproteases and serine proteases' contributions, proteolytic activity in the presence and absence of ethylenediaminetetraacetic acid (EDTA) and phenylmethylsulfonyl fluoride (PMSF) was assessed. The results showed a considerable increment of total proteolytic activity after venom injection in all organs assessed; moreover, EDTA presence reduced proteolytic activity more than by 50% almost in all assessed organs  and tissue inhibitors of matrix metalloproteases (TIMPs) was performed. The results showed a prominent increase in levels of collagenases , gelatinases , stromelysins , and TIMP-1 in all organs assessed; moreover, peak levels were observed in the 24 hours after venom injection, with further decrement of MMP and TIMP levels approximately to initial ones .Leiurus macroctenus envenomation were examined. As it turned out, Leiurus macroctenus envenomation causes significant elevations in proteolytic activity, mostly in the heart (3.34-fold increment) and lungs (2.25-fold increment). In the presence of EDTA, proteolytic activity was decreased by more than half in almost all organs, which indicates high metalloproteases content and activity during envenomation. In the presence of PMSF, proteolytic activity levels remained mostly above 60–70% in all organs, suggesting that serine proteases do not play a prominent role in proteolysis during envenomation. MMPs and TIMP-1 content assessments showed a considerable increase in levels of all MMPs and TIMP-1 in all organs assessed. Thus, the Leiurus macroctenus sting provides systemic envenomation, which may lead to multiple organ dysfunctions, mostly due to the uncontrolled metalloprotease activity.To sum up, total proteolytic activities as well as MMPs and TIMP-1 levels in the heart, lungs, intestine, liver, brain, kidneys, and spleen after As one of the most dangerous creatures on Earth, scorpions can cause huge problems when they encounter humans. It was previously reported that systemic envenomation effects are mostly caused by autonomic nervous system abnormalities . HoweverLeiurus macroctenus envenomation. Among all organs assessed, the heart and lungs appeared to have the highest increase in proteolytic activity after venom injection. Taking into account that cardiomyopathy and pulmonary edema are common symptoms of scorpion envenomation [Leiurus macroctenus envenomation.This study has revealed a significant increment in proteolytic activity levels during nomation –32, protLeiurus macroctenus venom is not an exception.According to the results obtained from the evaluation of metalloproteases and serine proteases' contributions to total proteolytic activity, metalloproteases were the major proteolytic agents in our experiment conditions, since EDTA treatment resulted in a reduction of the total proteolytic activity by more than half in most organs. A considerable decrement of proteolytic activity in EDTA presence during envenomation  indicates an increase in total proteolytic activity due to the increase in metalloproteases levels. In the presence of constant proteolytic activity levels and a parallel rise in total proteolytic activity, as was shown for the lungs, metalloproteases' absolute levels rose proportionally to total proteolytic activity, while their relative levels remained the same. In the brain, kidneys, and spleen, EDTA has not decreased total proteolytic activity compared to the control, proving that in these organs total proteolytic activity increased mainly due to the increment of other proteases' activities. PMSF treatment did not significantly reduce total proteolytic activity; it remained above 60–70% in almost all organs, even though in the heart, lungs, kidneys, and spleen it was lightly decreased during envenomation, suggesting that serine proteases are minor participants in tissue proteolysis processes during envenomation. With PMSF, higher proteolytic activity levels after venom injection, as compared to the control, indicate a decrease in relative serine proteases content during envenomation. Other authors report that metalloproteases, which are responsible for severe envenomation symptoms, are commonly present in relatively high concentrations in some jellyfish  and snakTaking into account the significant contribution of metalloproteases to the total proteolytic activity during envenomation, MMP and TIMP-1 levels were examined. The results showed a prominent increment of MMPs levels with a peak in 24 hours after venom injection in all organs assessed. Nevertheless, TIMP-1 levels were increasing simultaneously with MMP levels, thus, high TIMP-1 concentration decreased MMP levels in 72 hours after venom injection approximately to physiological values. The potential effects of MMP levels rising and their activation in tissues are varied, since there are several classes of MMPs based on their substrate type and structural properties . Even thLeiurus macroctenus sting may cause severe systemic envenomation since alterations in total proteolytic activity and MMP levels have been found in all organs assessed. The outcomes of envenomation may differ due to the large number of potential pathologies, which proteases, particularly metalloproteases, can trigger.Consequently, the


Results indicated the capability of UAE in extracting biologically active compounds from dill as a prospective functional material.This study evaluated the effect of ultrasonic-assisted extraction (UAE) on the isolation of phenolic compounds, flavonoids, and antioxidants from dill. UAE improved the extraction yields of total phenolic compounds and total flavonoid content as well as increased the antioxidant activities of all dill extracts. The optimum UAE condition to obtain highest total phenolic compounds, total flavonoid content, and antioxidant activities was 50% ethanol for 30 min giving 135.88 ± 3.23 mg gallic acid equivalent/g extract and 229.53 ± 4.97 mg rutin equivalent/g extract, respectively. Lowest IC Various secondary metabolites including phenolic compounds, flavonoids, carotenoids, terpenoids, and alkaloids have been found in plants at high concentration. These bioactive compounds are essential to the plant's defense mechanism and act as antioxidants, antimicrobials, and nutrients that can be used to treat and prevent a variety of diseases for humans. Specifically, the most abundant bioactive constituents identified in edible plants are flavonoids and phenolic compounds, which serve as non-enzymatic antioxidants in cells to prevent damage from oxidative stress, making them abundant sources of metabolites that support human health , 2.Examining extraction techniques is crucial for successfully obtaining these natural bioactive substances. Several methods for isolating natural bioactive compounds have been proposed. Conventional methods, such as Soxhlet extraction, reflux extraction, maceration, infusion, and percolation, are commonly used; however, these applications are time-consuming and involve high labor costs. Further, large amounts of extraction solvent are not environmentally friendly. Therefore, green alternative methods have been recently introduced to overcome these drawbacks; such techniques include ultrasonic-assisted extraction, microwave-assisted extraction, and supercritical fluid extraction. Ultrasonic-assisted extraction (UAE), among others, has enormous potential in the food and herbal industries because of its practicality and cost effectiveness. Additionally, it requires less time and energy, allows for low-temperature extraction, and maintains quality of the extract. UAE uses high-intensity sound waves to extract bioactive substances from their natural sources. Due to the acoustic cavitation generated by UAE, plant tissues are destroyed, and thus the contact area between solvent and plant materials is increased, enhancing higher extraction yield of bioactive compounds compared with conventional extraction methods –7.Anethum graveolens L.) is an important herbal plant belonging to the Apiaceae family; it is used as a local spice in Central and Southwest Asia, Southeast Europe, and the Mediterranean regions, although it can be grown around the world [This paper focuses on applying the UAE technique to extract phenolic compounds, flavonoids, and antioxidants from dill. Dill  at power and frequency of 350 W. and 50 Hz, respectively. Extraction variables of solvent concentration and time were investigated. Dried dill samples were extracted with ethanol , with sample: solvent ratio fixed at 1 : 10. Extraction times were set at 15 and 30 min. Liquid fractions were collected after centrifugation at 12,000 rpm for 10 min and concentrated by a rotary evaporator.To compare UAE with conventional methods (CE), the maceration method was employed to set up the conventional method. It was conducted in shaking incubator at 30°C and 150 rpm for 30 min and using 50% ethanol as extraction solvent.µL aliquot of dill extract was mixed with 500 µL of 10% Folin–Ciocalteu reagent and incubated at room temperature for 10 min. Then, 1 mL of 7.5% sodium carbonate was added, and the mixture was mixed and further incubated for 60 min at room temperature. Absorption at a wavelength of 765 nm was evaluated. TPC was expressed as mg gallic acid equivalent (GAE)/gram of extract by comparison with the gallic acid standard curve.The Folin–Ciocalteu method was used to determine TPC . A 20 µLµL aliquot of dill extract was mixed with 150 µL of 5% sodium nitrate and incubated at room temperature for 5 min. Then, 150 µL of 10% aluminum chloride was added. After 6 min incubation at room temperature, 500 µL sodium hydroxide was added to the mixture. Total volume was adjusted to 1.5 mL with 500 µL of distilled water. Absorption at a wavelength of 510 nm was evaluated. TFC was expressed as mg rutin equivalent (RE)/gram of extract by comparison with the rutin standard curve.Total flavonoid content was determined following the method of Wolfe et al. with some modifications . A 200 µc and Abss are the absorbances of the control (without dill extract) and sample (with dill extract), respectively. Linear regression of antioxidant activity was used to determine the concentration of dill extract that could reduce the concentration of ABTS radicals by 50% (IC50).ABTS  was used to determine the antioxidant activity of dill extracts following the method of Re et al. with some modifications . To genec and Abss are the absorbances of the control (without dill extract) and sample (with dill extract), respectively. Linear regression of antioxidant activity was used to determine the concentration of dill extract that could reduce the concentration of DPPH radicals by 50% (IC50).2,2-Diphenyl-1-picrylhydrazyl (DPPH) was used to determine the antioxidant activity of dill extracts according to the method described by Brand-Williams et al. with some modifications . An aliqα = 0.05.Data were expressed as mean ± standard deviation. Data with normal distribution were analyzed using one-way analysis of variance (ANOVA), followed by least significant difference (LSD) with a significance level of Total phenolic compounds and total flavonoid content of dill extracts are summarized in 50 values in 50 value indicates the concentration of dill extract that could reduce the concentration of ABTS or DPPH radicals by 50%. The IC50 results agreed well with the TPC and TFC results. Significantly lowest IC50 values for both ABTS and DPPH radicals were found from dill extracts obtained using 50% ethanol for 30 min of UAE as 0.034 ± 0.00 mg/mL and 0.12 ± 0.00 mg/mL, respectively, while a longer period of UAE did not cause negative feedback on antioxidant activities.The antioxidant activities of dill extracts against ABTS and DPPH radicals were also investigated and presented as IC50 value against ABTS and DPPH radicals of UAE was 0.034 ± 0.00 mg/mL and 0.12 ± 0.00 mg/mL, respectively, while the IC50 value against ABTS and DPPH radicals of CE was 4.43 ± 0.07 and 4.94 ± 0.03 mg/mL, respectively.To compare the efficiency of UAE with CE, samples obtained from CE (without ultrasonic treatment) were evaluated for their TPC, TFC, and the antioxidant activity. The results are presented in The biologically active compounds, including flavonoids, phenolic compounds, saponins, cardiac glycosides, and terpenes, have various health benefits and can be natural antioxidants that can neutralize free radicals and reactive oxygen species (ROS). Free radicals and ROS can attack biomolecules and healthy cells to lose their structures and functions, causing oxidative damage of DNA, proteins, lipids, and other biomolecules which lead to a variety of diseases. Thus, receiving the adequacy of these biologically active compounds, derived from potential plants either by direct consuming or dietary supplement, is the most effective way to prevent the oxidative damage of various biomolecules –13, 18. 50 reduced to 0.034 ± 0.00 mg/mL and 0.120 ± 0.00 mg/mL, respectively, which were about 130 and 41 times lower than those of CE. Comparing TPC, TFC, and antioxidant activities from the dill extract with UAE treatment to those of other plant species, they were considerable. In the study by Saeed et al., TPC and TFC were evaluated from an extract of bristle fruit hedge parsley (Torilis leptophylla), which belongs to the same family as dill, the Apiaceae family. It was extracted by methanol via maceration technique for 2 days. The TPC and TFC of the methanol extract were 121.9 mg GAE/g extract and 59.6 mg RE/g extract, respectively, according to their research. In addition, it had IC50 values of 0.189 and 0.179 mg/mL for scavenging the ABTS and DPPH radicals, respectively [According to our study, the edible plant portions of dill were a good source of phenolic compounds, flavonoids, and antioxidants. UAE greatly increased extraction efficiency and generated dill extracts with very highly active ingredients. For dill extracts, using 50% ethanol for 30 min was the optimum UAE condition. Yields of TPC and TFC, measuring as 135.88 ± 3.23 mg GAE/g extract and 229.53 ± 4.97 mg RE/g extract, respectively, were found to be approximately 50 and 69 times higher than those of CE. The scavenging ability against ABTS and DPPH radicals significantly increased while extracting under UAE conditions; the ICectively .The improvement in TPC, TFC, and antioxidant activities of dill extracts was attributed to the pervading nature of the ultrasonic waves that produced unstable cavitation bubbles which formed high-velocity jets upon collapsing . This caOur results indicated that the UAE efficiency of dill extract was influenced by both solvent concentration and extraction time. In this study, ethanol and water were chosen as the extraction solvents due to safety and health considerations. Our findings were consistent with earlier studies, which indicated that employing a binary solvent system was preferable to a monosolvent system –27. UsinExtension of UAE time to 30 min did not cause oxidation of TPC, TFC, and antioxidants. This result was in line with earlier research which found that TPC and TFC extraction efficiency was increased by longer UAE times, whereas an extension of the UAE treatment to 40–60 min could have negative effects by enabling the active compounds to oxidize and get destroyed , 26, 28.To assess the free radical scavenging capacity of diverse substances, the ABTS and DPPH radical scavenging capacity assay is widely utilized. The ABTS and DPPH assays showed the increasing scavenging activity trends when the extraction time increased at all ethanol concentration. In this study, 50% ethanol and 30 min condition were considered the most effective way for dill extraction. This situation could be explained by the fact that the solubility of antioxidants depends on the chemical structures and the polarity of the solvent system. The estimation of ABTS and DPPH radical scavenging capacities is based on the capacity of existing compounds to scavenge ABTS and DPPH radicals. Our findings demonstrated the influence of solvent selection on the effectiveness of antioxidant extraction. It was possible that antioxidants, which are less hydrophilic, could not be extracted using pure water, or in other words, using 0% ethanol, while more hydrophilic antioxidants were more easily released when there is water present during the extraction. Therefore, using a mixture of ethanol and water would be more appropriate in isolating a wide range of antioxidants from dill.However, applying UAE to extract active compounds from nature was not successful for all cases. In the study by Ramli et al. for instance, it was discovered that UAE dramatically increased the flavonoid content and scavenging activity for the peel of red dragon fruit while significantly decreasing the extraction yield. UAE, on the other hand, boosted extraction yield while lowering flavonoid concentration and scavenging activity for flesh of red dragon fruit . This de50 values against ABTS and DPPH radicals as 0.034 ± 0.00 mg/mL and 0.12 ± 0.00 mg/mL, respectively.Our investigations indicated that UAE assisted the extraction process of TPC, TFC, and antioxidants from dill. The optimum UAE condition for dill extraction was 50% ethanol and 30 min. Highest yields of TPC and TFC were 135.88 ± 3.23 mg GAE/g extract and 229.53 ± 4.97 mg RE/g extract, respectively, with lowest IC


Most studies have focused on large scale computational predictions of de novo protein‐coding genes across a wide range of organisms. In contrast, experimental data concerning the folding and function of de novo proteins are scarce. This might be due to difficulties in handling de novo proteins in vitro, as most are short and predicted to be disordered. Here, we propose a guideline for the effective expression of eukaryotic de novo proteins in Escherichia coli. We used 11 sequences from Drosophila melanogaster and 10 from Homo sapiens, that are predicted de novo proteins from former studies, for heterologous expression. The candidate de novo proteins have varying secondary structure and disorder content. Using multiple combinations of purification tags, E. coli expression strains, and chaperone systems, we were able to increase the number of solubly expressed putative de novo proteins from 30% to 62%. Our findings indicate that the best combination for expressing putative de novo proteins in E. coli is a GST‐tag with T7 Express cells and co‐expressed chaperones. We found that, overall, proteins with higher predicted disorder were easier to express.Over the past decade, evidence has accumulated that new protein‐coding genes can emerge de novo proteins. Their properties are still poorly understood and their experimental analysis faces major obstacles. Here, we aim to present a starting point for soluble expression of de novo proteins with the help of chaperones and thereby enable further characterization.Today, we know that proteins do not only evolve by duplication and divergence of existing proteins but also arise from previously non‐coding DNA. These proteins are called  Due to their recent emergence, de novo genes tend to be shorter, evolve more rapidly, and have lower expression than established genes.de novo genes. By combining homology and synteny based approaches for de novo gene identification, the origin of de novo genes can be detected more accurately.de novo protein‐coding genes have been identified and confirmed across a wide range of eukaryotes.de novo genes were mainly analyzed with comparative genomics and transcriptomics. A recent study by Grandchamp et al.de novo gene emergence,de novo proteins emerge, remains incomplete.Several de novo proteins, together with the novelty of the research area, might be the reason for the lack of experimental studies on de novo proteins. So far only two de novo proteins were expressed and characterized experimentally, Goddard (Gdrd)de novo protein was difficult to analyze due to unstable or incorrect folding (Bsc4) or unusual behavior in SDS‐PAGE (Gdrd). Compared to well‐studied proteins with expression and purification data available, de novo proteins tend to behave differently when using standard protocols.Difficulties in handling de novo proteins. Since de novo proteins evolve rapidly by becoming coding from scratch, they probably lack a stable structural configuration and contain high amounts of disorder.in vitro experiments and could explain the obstacles faced during their expression.de novo proteins undergo a similar hindrance in their native organism or only in the expression hosts.de novo protein expression might also profit from chaperones. With their “emergence from dark genomic matter” in the DNAde novo proteins are prospective targets for chaperones because their solubility can be increased.Several studies, foremost some from the laboratory of Dan Tawfik,The chaperonin GroEL and its co‐chaperone GroES are found throughout the bacterial domain, while their homologs, HSP60 and HSP10, respectively, are found in eukaryotes.The other chaperone system used here is DnaK, DnaJ, and GrpE (homologous to HSP70 and HSP40 in eukaryotes). For simplicity we will refer to the chaperone system GroEL/ GroES as only GroEL and to DnaK, DnaJ, and GrpE as DnaK only. While the GroEL system targets misfolded and unfolded proteins, DnaK can refold an already aggregated protein to its native state using ATP  and 10 from Homo sapiens (termed here as HS1‐10) as shown in Figure de novo proteins can be found on Zenodo (https://doi.org/10.5281/zenodo.6512224), for genomic location and official gene names see Table de novo proteins have been recently published by Heames et al.de novo protein from D. melanogaster, called Atlas. Atlas appears to function as a DNA binding protein that facilitates the packaging of chromatin in developing D. melanogaster sperm.de novo proteins is still underrepresented  and challenging, we want to propose a guideline for successful expression of putative de novo proteins in E. coli. We combined different chaperone systems (GroEL and DnaK) with different combinations of E. coli strains (BL21 Star™ [DE3] and T7 Express) in order to express putative de novo proteins solubly. To verify successful expression of target proteins, Western blots were performed and samples sent for tryptic digest followed by mass spectrometry. We identified the best combination for expression of putative de novo proteins in E. coli. After first expressions with His‐tag alone resulted in soluble expression for only 1/21 proteins, we increased the total number of solubly expressed putative de novo proteins to 13/21 with GST‐tag and chaperones. The different chaperone systems increased or enabled soluble expression in four cases, while DnaK only helped in two, GroEL in all of those four.For this study, we used 21 putative 22.12.1.1de novo proteins. For this we calculated the percentage of residues predicted to be disordered , DM2 , DM4 , DM5 , and DM10  are predicted to have secondary structure contents of 50% or more, with α‐helices to be more frequent than β‐sheets and less than 50% random coils. HS1 , HS3 , HS4 , HS5 , HS6 , HS7 , DM3 , DM7 , and DM9 , on the other hand, are predicted to be mostly random coils (disordered) with otherwise higher amounts of β‐sheets predicted. DM6 (∼90% random coils), DM8 (∼100% random coils), HS2 (∼85% random coils), HS9 (∼95% random coils), and HS10 (∼100% random coils) are predicted to contain more or less only random coils.Our goal was to choose a cohort of 2.22.2.1de novo protein Gdrd.11 putative de novo protein candidates with an N‐terminal 6xHis‐tag in E. coli BL21 Star™ (DE3) cells, and verify expression via SDS‐PAGE and mass spectrometry. However, for our candidates, the expression level was either very low or not detectable, as can be seen in Figure E. coli cells (T7 Express), but expression remained unsuccessful. Shifting from an N‐terminal 6xHis‐Tag to a C‐terminal 6xHis‐tag showed similar negative results. Considering the size and levels of disorder, we switched to a larger tag for increased solubility and stability, choosing an N‐terminal GST‐tag. In this way, we were able to observe a higher success rate in soluble expression of our target proteins. But not all proteins could be expressed at satisfying levels, especially solubility needed to be increased for some  and T7 Express E. coli cells. Six candidate proteins were expressed in T7, two were expressed in BL21 Star™ (DE3) cells. Three proteins were not expressable in either strain. In summary, with the combination of chaperones and switching to N‐terminal GST‐tag, we were able to express 8/11 of the D. melanogaster putative de novo protein candidates  without chaperones (base), (ii) with GroEL, and (iii) with DnaK. Surprisingly, we did not observe increased solubility for most putative DM1, DM2, DM3, DM4, and DM7. In combination with GroEL, the intensity of the bands in the soluble fraction and therefore amount of soluble protein, even decreased for DM3, DM4, and DM7. For DM2 and DM5, the amount of soluble protein increased when co‐expressed with GroEL. When DnaK was co‐expressed, protein solubility either appeared to decrease  or was similar to the base (DM3 and DM7). DM8 showed similar soluble expression for all three conditions with most of the protein being insoluble. In the case of Atlas and DM5, soluble protein expression was increased or enabled with the addition of the GroEL chaperone system while DnaK and base expression resulted in no or very little soluble protein. While we cannot confirm that co‐expression with DnaK in fact decreases the amount of soluble protein , we do not see increased soluble expression for any of the candidate proteins in the presence of DnaK as we do for GroEL (DM5 and Atlas).In contrast, we observed soluble expression for most proteins without chaperones, for example, 2.2.3de novo proteins were expressed following the same protocol as the D. melanogaster proteins by combining the different E. coli expression cells, tags, and chaperone systems , however, was only weakly expressed with an N‐terminal 6xHis‐tag but using also E. coli T7 express cells. Without the addition of chaperones only HS7, HS8, and HS10 were successfully expressed and soluble. After co‐expression with chaperones, as described for D. melanogaster proteins, two more H. sapiens proteins could be expressed. Unfortunately, H. sapiens protein candidates HS1, HS3, HS4, HS5, and HS6 showed no expression at all, even with chaperones. In total, we were able to express 5 out of 10 putative de novo proteins following our protocol  showed very weak or no soluble expression without chaperones, but solubility could be increased with both chaperone systems. HS8 and HS10 showed low soluble expression overall, but no change in solubility was visible when co‐expressing with either chaperone system. The candidate de novo protein HS7 already showed strong soluble expression at base  base, (ii) GroEL, and (iii) DnaK, as described above. Two out of the five successful candidates : BL21 Star™ and T7 Express. Both strains resulted in effective protein expression and a relatively high yield of the de novo proteins, with T7 Express being the best option. The de novo proteins studied here are possibly a toxic, metabolic burden to the E. coli cells, suggesting T7 cells are the better choice of expression cell. BL21 Star™ (DE3) contains a T7‐RNA‐polymerase under control of lacUV5 promoter together with higher mRNA stability. This leads to stable mRNA transcripts and higher amount of target protein. However, BL21 Star™ (DE3) cells have increased basal expression of heterologous genes and cannot express toxic genes. In contrast, the T7 Express cells have a reduced basal expression of target proteins than BL21 Star™ (DE3) cells. Therefore, toxic proteins can be expressed better in T7 cells compared with BL21 Star™ (New England Biolabs).3.2de novo protein candidates are quite small (8–12 kDa), so a larger tag like GST might already stabilize in a chaperone‐like manner.HS8, that is, 2/21, were only expressed with an N‐terminal 6xHis‐tag. With a mass of only 1 kDa, 6xHis‐tag is the better choice for further structural characterization using circular dichroism (CD), multi‐angle light scattering (MALS) or nuclear magnetic resonance (NMR), since a small tag has less influence on protein folding. In contrast, the larger GST‐tag needs to be cleaved for most follow‐up experiments. When removing the tag, the de novo protein might behave differently and could degrade or aggregate.Based on our study, an N‐terminal GST‐tag was the more appropriate choice than a 6xHis‐tag. Some 3.3HS2 and HS9, 2/21) but for most proteins GroEL leads to more soluble protein than DnaK   in a cell‐free expression system.Our Western blot results indicate that GroEL slightly outperforms DnaK in terms of increased protein solubility. In some cases, both chaperone systems increase or enable soluble expression  Figure . DnaK reDM1, DM2, HS7, and Atlas), DnaK decreased the solubility of proteins that expressed soluble without chaperones. One could assume that DnaK and target protein expression compete for cellular ressources and that for already soluble proteins this has no positive trade‐off. Another reason could be DnaK's role as central organizer in the chaperone network of E. coli. Binding to DnaK could trigger degradation of the “toxic” target protein but also overexpression of DnaK additionally to the endogenous version could bring the cellular metabolism out of balance.In some cases . For both, D. melanogaster and H. sapiens candidate putative de novo proteins, the combination of GST‐tag and E. coli T7 Express cells were the best performing (10 out of 13). We performed test expressions and compared the levels of soluble expression for different chaperone combinations shown in Figures de novo protein candidates DM5, Atlas, HS2, and HS9 were in line with our original hypothesis that chaperones enhance solubility of de novo proteins in heterologous expression systems. However, the choice of appropriate tag and expression cells in the first step was equally, if not more, important. When using the N‐terminal His‐tag that proved successful for putative de novo protein Gdrd, only two (Atlas and HS7) of our candidate proteins were expressed. When switching to the N‐terminal GST‐tag another seven D. melanogaster and four more H. sapiens protein candidates were expressed. Unfortunately, we were not able to express 8/21 of the candidate proteins in E. coli at all , despite trying different expression strains, tags and chaperone systems.In total, we were able to successfully express 13 out of 21 putative 3.5de novo protein candidates, we observe a slight trend toward better expression of the more disordered proteins. This trend can be observed for the IUPred2a disorder predictions  which are predicted to contain over 70% random coils (or 60% disorder). The described differences in predicted secondary structure content and disorder level might be the reason why these putative de novo candidates could not be expressed in E. coli cells even with the help of chaperones.When examining the predicted structural properties of the human s Figure . The unsD. melanogaster protein candidates, this trend was not observed. Here, several of the proteins with lower disorder predicted  were expressed solubly without addition of chaperones. Yet, DM6 (∼90% disorder predicted) was not expressed successfully. However, the two proteins with 100% random coils predicted by Porter 5.0 and highest disorder predictions by IUPred2a (DM8 and HS10) did not show any change in solubility when chaperones were co‐expressed. Considering that such highly disordered proteins do not need chaperones, this observation was expected.For HS9 and DM3) while not invalidating one another.Deviations of the level of predicted disorder and predicted secondary structures, especially random coils, for each protein can be explained by the differences in IUPred2a and Porter 5.0. IUPred2a provides energy estimations for each amino acid residue resulting in quasi‐probabilities of disorder.4de novo protein expression is only the first step toward enabling further in vitro experiments for functional and structural characterization. Further advancement will lead to efficient and stable purification, followed by functional assays such as peptide phage display to identify binding partners.de novo proteins experimentally. Even in light of the recent dawn of computational structure prediction,de novo proteins. While contemporary prediction methods can certainly provide a first estimate on structure, the intrinsic nature of de novo proteins, with their short length, high disorder content and lack of homology, will demand some scepticism while analyzing such predictions.de novo proteins, one could assume that de novo proteins are of such small size that AlphaFold can solve their local folding. This will have to be validated in future studies. This study of 21 putative de novo proteins from H. sapiens and D. melanogaster, including previously in vivo characterized putative de novo protein Atlas, showed that chaperones may help expressing de novo proteins in E. coli cells. However, not all putative de novo proteins needed chaperones for soluble expression and sometimes even expressed better without. Fusion of the target de novo proteins to a GST‐tag and using T7 Express cells as hosts proved to be the most successful combination. Our work may serve as a guide to facilitating future analyses of putative de novo proteins or other difficult (short and/or disordered) target proteins in E. coli.Exemplifying the general trend for soluble 55.1https://doi.org/10.5281/zenodo.6512224).All SDS‐PAGEs, MS results, Western blots, and scripts are deposited in Zenodo database  and dividing it by the sequence length of the protein. We also predicted average disorder and percentage of disordered residues with a disorder threshold of 0.8  inside the sequence. The wild‐type DNA for Atlas was provided by Geoff Findlay. To introduce restriction sites at the ends, we used different primers (a fasta file containing the DNA sequences and primer used can be found online on Zenodo (https://doi.org/10.5281/zenodo.6512224) For cloning into pHAT2 vector  we used restriction enzymes combination of BamHI/XhoI + HindIII, for pETM‐30 , we used NcoI+HindIII. Both vectors were from the EMBL vector database, Heidelberg, introduced stop‐codon was TAA for all constructs. We digested the PCR product with both restriction enzymes respectively  for 3 h at 37°C. Digest of the vector  was purified from agarose gel (Zymo Research). We ligated both with an insert:vector ratio of 1:4 using Ligase . The ligation mix was purified (Zymo Research) and 2 μl of the purified reaction mix was used to transform into 50 μl of chemically competent E. coli TOP10 cells. Cells were incubated for 30 min on ice, followed by a 90 sec heat‐shock at 42°C. 500 μl of LB‐Media  was added for recovery and incubated for 1 h at 37°C. After incubation, the resuspended cell pellets were plated on LB‐agar containing 50 μg/ml ampicillin  or Kanamycin  and incubated at 37°C over night.Putative E. coli Top10 cells and then into expression strains (BL21 Star™ (DE3) and T7 Express). Chaperone plasmid bearing cells were made chemically competent (Inoue method)Successful transformation was verified by colony PCR and sequencing at Microsynth, Seqlab, Germany. The plasmid DNA bearing the chaperone combinations GroEL/ES (pGro7) or DnaK/J/GrpE (pKJE) from Takara Biotech chaperone kit5.3.2de novo proteins.To identify in which strain and plasmid proteins were expressed we performed test expressions. 10 ml of LB + AMP + CAM or LB + KAN + CAM were inoculated from a glycerol stock of all three expression cells bearing both plasmids (target protein and chaperone) and grown until turbid . We split the solutions into 3 × 3 ml and incubated for 30 min at different temperatures  before adding IPTG (Carl Roth) for a final concentration of 0.5 mM and shaking over night. When using the cells with chaperone plasmids we made the following adjustment: L‐arabinose  was added from the beginning for immediate induction of chaperone expression. Therefore, after inducing the de novo protein expression with IPTG the chaperones were already present in order to help folding the A total of 500 μL of each cell culture were centrifuged . Pellets were resuspended and lyzed in 50 μl of a mix of Bugbuster and Lysonase (both Merck AG) through vortexing for 10 min. After centrifugation the supernatant was mixed with the same volume of SDS‐loading buffer (standard). The pellet was resuspended in 5x diluted Bugbuster, centrifuged, and resuspended in 50 μl SDS‐loading buffer. 15 μl of each fraction was loaded on an SDS‐PAGE, either 10% Bis‐Tris or 12.5% TGS, run on 200 V for 50 min and dyed using ReadyBlue™ staining.For the final Western blots, the determined optimal combination of strain, expression vector, and chaperone plasmid were used. 20 ml cultures of 2YT + AMP + CAM or 2YT + KAN + CAM were inoculated with 1 ml of the overnight culture. L‐arabinose  was added to the samples, but not to the control without chaperones and grown at 37°C, 180 rpm for 4–6 hr until turbid. The cultures were incubated at 28°C, 180 rpm for 30 min before induction with IPTG  and incubated overnight under these conditions. Final samples were harvested and handled as prior performed test expressions.5.3.3The SDS‐PAGEs were run as described above but without ReadyBlue™staining. The gel was equilibrated in transfer buffer (20% Methanol) for a few seconds. A polyvinylidene fluoride (PVDF) membrane with a pore size of 0.22 μm was activated by methanol (2 min) and equilibrated in transfer buffer. The semi‐dry transfer was performed at 25 V for 30 min using the BioRad standard protocol. The membrane was blocked at room temperature for 1 hr using 5% bovine serum albumin BSA in phosphate‐buffered saline with tween (PBS‐T) then washed in PBS‐T and incubated for 1 h with anti‐His antibody (MA1‐21315‐HRP) diluted 1:500. For chemiluminescence, 0.5 ml luminol was mixed with 0.5 ml peroxide and distributed evenly on the membrane. Intensities of the different bands were measured in ImageJ after default background subtraction. The different fractions  are calculated as fractions of the intensities of all relevant protein bands to also compare the amount of expression between the different conditions.5.3.4Tryptic digest followed by mass spectrometry for peptide detection of the candidate proteins was performed by the Core Unit Proteomics group of Prof. Dr. Simone König, UKM Muenster.Lars A. Eicholt: Data curation (lead); investigation (lead); validation (lead); visualization (lead); writing – original draft (lead). Margaux Aubel: Data curation ; investigation ; validation ; visualization ; writing – original draft . Katrin Berk: Data curation (supporting); validation (supporting); writing – original draft (supporting). Erich Bornberg‐Bauer: Conceptualization (supporting); funding acquisition (lead); project administration (supporting); resources (lead); supervision (supporting); writing – original draft (supporting). Andreas Lange: Conceptualization (lead); investigation (supporting); project administration (lead); supervision (lead); validation (supporting); writing – original draft (supporting).The authors declare no competing interests.Figure S1 Percentage of average disorder as calculated with IUPred2a. All candidate de novo proteins used for expression experiments ordered by their average disorder level from left to right.Click here for additional data file.Figure S2 Number of DnaK binding Heptamers as predicted with ChaperISM suite (v1) per candidate de novo protein. DM3 was expressed solubly with DnaK, but no binding sites were predicted.Click here for additional data file.Figure S3 SDS‐PAGEs with all D. melanogaser de novo protein candidates co‐expressed with GroEL system and without. The red boxes indicate GroEL chaperone, green boxes the target protein.Click here for additional data file.Figure S4 SDS‐PAGEs with solubly expressed de novo protein candidates of H. sapiens co‐expressed with GroEL system and without. The red boxes indicate GroEL chaperone, green boxes the target protein.Click here for additional data file.Table S1 Names of the putative de novo proteins, annotated gene names (only for D. melanogaster) and genomic locations on genome assembly BDGP6.32 (D. melanogaster) and GRCh38.p13 (H. sapiens).Click here for additional data file.