To study the effect of carbetocin on intraoperative blood loss and thickness of myometrium during cesarean section with the scarred uterus at term pregnancy.  Pregnant women with full-term gestational scar uterus who underwent cesarean section from March 1, 2021, to April 30, 2022, were retrospectively collected and divided into a reference group (using oxytocin) or a study group (using carbetocin). The clinical data of the two groups were retrospectively analyzed, and the operation time, intraoperative blood loss, hospital stay, uterine contraction effect, changes in the myometrium, and complications were compared between the two groups. p=0.0001, 0.005, 0.006, 0.001, 0.0004, 0.003, 0.001, and 0.005, respectively). There were no significant differences in estradiol (E2), luteinizing hormone (LH), or follicle-stimulating hormone (FSH) between the two groups before the surgery , but there were significant differences after the surgery .  A total of 103 pregnant women were retrieved. There were 44 cases in the reference group and 59 cases in the study group. There were significant differences in operation time, intraoperative bleeding, hospital stay, postoperative adverse events, uterine fundus wall thickness, anterior wall thickness, posterior wall thickness, and uterine contraction effect between the two groups ( The use of carbetocin in the cesarean section of a full-term scar uterus is significantly effective in shortening the operation time, reducing the amount of intraoperative blood loss, and promoting the recovery of the uterus. The scar in the uterine wall area after cesarean section or myomectomy is defined as the uterine scar. A second cesarean section is generally performed after the scarred uterus is pregnant, because the elasticity of the muscle fibers in the scar area located in the lower uterus will be significantly reduced, which will lead to weak uterine contractions, resulting in postpartum hemorrhage. At the same time, the scar will also cause certain negative effects on the recovery of the uterus. This leads to endometrial and pelvic infections, which aggravate postpartum morbidity and pain in mothers. Therefore, drugs that can increase uterine contractions should be used to reduce the risk of postpartum hemorrhage –3. TherePregnant women with full-term gestational scar uterus who underwent cesarean section from March 1, 2021, to April 30, 2022, were retrospectively collected and divided into a reference group (using oxytocin) or a study group (using carbetocin). Inclusion criteria were as follows: full-term pregnancy; scarred uterus; cesarean section required; and no related treatment was performed prior to inclusion in the study. Exclusion criteria were as follows: coagulation dysfunction; malignant tumors; chronic diseases such as diabetes, pregnancy-induced hypertension, etc.; mental illness; or allergic to drugs.The clinical data of the two groups were retrospectively collected, and the operation time, intraoperative blood loss, hospital stay, uterine contraction effect, changes in the myometrium, and complications were compared between the two groups. All patients in this study gave informed consent. This study was approved by the institutional ethical committee of our hospital.All cases underwent cesarean section of the lower uterine segment after spinal anesthesia. The control group was given 100 micrograms of oxytocin, and the study group was given 100 micrograms of carbetocin during c-section.2 (%) tests were performed for the count data, and the t-test (x ± s) tests were performed for the measurement data. A two-sidedp < 0.05 was determined as the threshold of statistical significance.Data were analyzed by SPSS21.0 , in which the There was no significant difference in clinical characteristics between the two groups .p=0.0001, 0.005, and 0.006, respectively). See There were significant differences in operation time (82.18 ± 10.01 vs 104.28 ± 10.33), intraoperative bleeding (210.36 ± 25.89 vs 316.27 ± 20.57), hospital stay (5.86 ± 2.51 vs 7.86 ± 2.57) between the study group and the reference group (p=0.001). See There was a significant difference in the incidence of postoperative adverse events between the study group and the control group . See There were significant differences in uterine fundus wall thickness (9.18 ± 1.01 vs 7.28 ± 1.33), anterior wall thickness (8.36 ± 1.89 vs 6.27 ± 1.57), and posterior wall thickness (8.86 ± 1.51 vs 6.86 ± 1.57) between the study group and the control group  and the reference group . However, after c-section, there were significant differences in E2 (60.96 ± 12.35 vs 66.37 ± 10.07), LH (7.65 ± 2.21 vs 9.25 ± 3.35), FSH (2.08 ± 0.79 vs 3.59 ± 0.51) between the study group and the control group , luteinizing hormone , or follicle-stimulating hormone  between the study group and the reference group (In recent years, due to the continuous development of anesthesia and cesarean section technology, the incidence of cesarean section in China has been as high as 80% in some places. With the implementation of two- and three-child policies, the cicatricial uterus is more common in obstetrics . EvidencBecause uterine atony is an important factor leading to massive postpartum hemorrhage, oxytocin-family drugs are used in the routine cesarean section to decrease the risk of postpartum hemorrhage. Oxytocin works quickly, but its half-life is generally 3–4 minutes, requiring multiple or continuous applications. When the dose exceeds 60 U, there will be obvious receptor saturation, which significantly weakens the effect on the lower uterine muscle group. In addition, taking large doses of oxytocin can also cause side effects such as hypotension and arrhythmia. Our study showed that carbetocin has a good hemostatic effect, and the effect is rapid and long-lasting –10. ThisBecause fibrous scars significantly reduce the contraction and elongation of the uterus, the scarred uterus is likely to have uterine rupture during pregnancy, vaginal delivery is not recommended. Cesarean section is a common gynecological operation, and its surgical methods and procedures have become mature . But posTo sum up, the use of carbetocin in the cesarean section of the full-term scarred uterus has a significant effect, which can effectively shorten the operation time, reduce the amount of intraoperative blood loss, and promote the recovery of the thickness of the anterior and posterior uterine walls and the myometrium, with fewer adverse events.


CHAMP1-related neurodevelopmental disorder, or CHAMP1 disorder, is a recently described genetic syndrome associated with developmental delay, intellectual disability, behavioral symptoms, medical comorbidities, and dysmorphic features. To date, literature has focused on medical review and dysmorphology but has yet to prospectively assess neurobehavioral core domains such as autism, or behavioral, language, cognitive, and sensory features. Here, we present deep phenotyping results for 11 individuals with CHAMP1 disorder, based on approximately 12 hours of remote clinician-administered assessments and standardized caregiver questionnaires. Diagnoses of autism spectrum disorder were given to 33% of participants; repetitive behaviors and sensory-seeking symptoms were prominent in this cohort. In addition, 60% of participants met the criteria for attention-deficit/hyperactivity disorder (ADHD). High rates of ADHD and relatively low rates of treatment suggest potential areas for intervention. This study represents the first prospective phenotyping analysis of individuals with CHAMP1 disorder. The utility of specific measures as clinical endpoints, as well as benefits and limitations of remote phenotyping, are described. NLGN3, SHANK3), chromatin remodeling , transcriptional regulation  . CHAMP1, a gene involved in proper chromosome segregation, has recently been associated with a neurodevelopmental disorder  ,2. The d, FOXP1)  and chrodisorder ,19.CHAMP1 pathogenic variants were first identified in 2 of 1133 children with severe DD in the Deciphering Developmental Disorders study  , and sleep disturbance  . All exhturbance . In 2016entation . The lardisorder , adding CHAMP1 remains a rare cause of NDDs, possibly accounting for 0.03% of NDD cases. Clinical laboratories have seen positive CHAMP1 findings for 22 of 6,2 586 and 5 of 1,1 000 exomes at GeneDX and Ambry Genetics, respectively  and began to walk between 18 and 48 months (26.6 ± 9.4) . All parNine of 11 participants had a minimum of single-word speech at the time of evaluation. Language milestones were delayed in all participants. First word was achieved between 18 and 84 months (41.8 ± 23.2). Only four participants had phrase speech, which developed between 58 and 180 months (101.5 ± 57.21) . ParticiOverall, there was minimal evidence of regression in this cohort. Minor reported regressions included two individuals who lost babbling, one at 10 months and the other at 14 months; participants regained this skill at 16 and 24 months, respectively. Additionally, there was one participant who lost the ability to roll over at 11 months; this skill was regained at 22 months. Notably, there was no reported regression in social skills , motor skills other than rolling over , or language skills other than babbling .The DP-4 Cognitive domain standard scores ranged from 40 (floor) to 85 (49.8 ± 15.8) . The youn = 5), with scores ranging from 52 to 65 (57.8 ± 5.6). Subdomain scores are shown in Adaptive functioning was significantly affected in all participants. Scores on the Vineland-3 Adaptive Behavior Composite ranged from 20 (floor) to 70 (44.0 ± 17.0), where the youngest participant had the highest score. Comparing each domain, scores were the highest in the Socialization domain, which ranged from 20 to 85 (48.0 ± 21.6) and lowest in the Communication domain, which ranged from 20 to 66 (37.7 ± 16.0). Scores in the Daily Living Skills domain ranged from 20 to 61 (44.0 ± 16.6). The Motor domain was completed in individuals of age 7 years and younger (obs), three individuals met for severe ASD, two for mild–moderate ASD, and five did not surpass the threshold for ASD. On the psychiatric evaluation, 9 of 11 participants presented with at least one symptom of ASD. Overall, three participants met for a consensus Diagnostic and Statistical Manual for Mental Disorders, Fifth Edition (DSM-5) diagnosis of ASD. Specific DSM-5 criteria for each individual can be found in obs had 38% true positives, 25% false positives, and 38% true negatives; there were no false negatives when compared to consensus diagnosis.Seven of 10 participants surpassed the cutoff on all the Autism Diagnostic Interview-Revised (ADI-R) domains. All 10 met the cutoff in the Abnormality of Development domain, 9 in Restricted and Repetitive Behavior (RRB), and 8 in Abnormalities in Reciprocal Social Interaction domain. In the Communication domain, four out of four met criteria on the Verbal algorithm and five out of six on the Nonverbal algorithm. On the Childhood Autism Rating Scale , 5 of 10 participants’ Total T-scores fell in the severe range, three in the moderate range and two in the normal range, with an average Total T-score of 73.5 ± 11.4. Five participants scored in the severe range in the Social Cognition domain (70.7 ± 13.6), four in the Social Motivation domain (67.1 ± 11.8), two in the Social Communication domain (68.6 ± 11.8), and one in the Social Awareness domain (61.1 ± 8.7). Eight of 10 participants scored in the severe range in the RRB domain; overall, the cohort had an average RRB T-score of 82.2 ± 13.4.The total score on the Repetitive Behavior Scale-Revised (RBS-R) ranged from 2 to 71 (29.4 ± 19.8), with a total maximum score of 129. The cohort had the highest scores, proportionally, on the Ritualistic domain, scoring on average 6.0 ± 4.4 out of 18 total points. The next highest scores were within the Restricted Behavior and Sameness Behavior domains, where participants scored 3.6 ± 2.9 of 12 total points and 8.0 ± 6.3 of 33 total points, respectively. The cohort scored the lowest on the Stereotyped Behavior, Compulsive Behavior, and Self-Injury domains with 3.6 ± 2.4 of 18, 4.3 ± 4.0 of 24 and 3.7 ± 4.4 of 24 points.N = 54, Mage = 5.4), individuals with CHAMP1 disorder had significantly more sensory features based on SAND interview scores in total and on every subdomain (P < 0.01). In the typically developing cohort, the average reported total score was 2.7 ± 2.3, reported hyperreactivity was 1.3 ± 1.5, hyporeactivity was 0.3 ± 0.7, and seeking was 1.1 ± 1.5. The average reported visual score in the typically developing cohort was 0.4 ± 0.9, reported tactile was 1.2 ± 1.8, and reported auditory was 1.1 ± 1.4.The Sensory Assessment for Neurodevelopmental Disorders (SAND) Total Reported scores ranged from 6 to 33 (20.1 ± 9.3). Additionally, results indicated that participants had the most sensory seeking symptoms (7.8 ± 4.3), followed by hyporeactivity (6.3 ± 3.1) and hyperreactivity (6.0 ± 4.2) symptoms. In terms of sensory modalities, participants showed the most tactile symptoms (9.0 ± 2.5), followed by visual symptoms (5.8 ± 3.8) and auditory symptoms (5.3 ± 4.6). Compared to a typically developing cohort . In the Quadrant scores, all nine individuals had definite differences in Low Registration, seven in both Sensation Seeking and Sensory Sensitivity, and six in Sensation Avoiding. Caregivers reported the highest sensory behaviors on the SP in Touch Processing, where 9 of 9 individuals scored in the definite difference range. This was followed by Multisensory Processing, where eight individuals had definite differences; Vestibular Processing, where six individuals had definite differences; and Auditory and Visual Processing, where three individuals each had definite differences.On the Sensory Experiences Questionnaire Version 3.0, participants scored highest, on average, in the Sensory Interests, Repetitions and Seeking Behavior domain, with an item mean of 2.5 (0.4) of 5. They scored higher in the Hyperresponsiveness domain (2.3 ± 0.6) than Hyporesponsiveness (2.2 ± 0.3), and they scored the lowest in the Enhanced Perception domain (2.0 ± 0.6). Participants demonstrated increased sensory symptoms in the Social Context (2.4 ± 0.4) compared to the Non-social Context (2.3 ± 0.4). Comparing sensory modalities, the cohort exhibited the highest scores in the Tactile domain (2.6 ± 0.3), followed by the Visual domain (2.3 ± 0.4), Vestibular/Proprioception domain (2.2 ± 0.4), Auditory domain (2.2 ± 0.6), and the Gustatory/Olfactory domain (2.2 ± 0.3).Standard scores on the Peabody Picture Vocabulary Test, Fifth Edition (PPVT-5) ranged from 40 to 83 (56.6 ± 12.3), and 40 to 72 (46.2 ± 11.0) on the Expressive Vocabulary Test, Third Edition (EVT-3) . Four inScores on the Vineland-3 Receptive Language subdomain ranged from 1 to 10 (5.9 ± 4.1), 1 to 7 on the Expressive Language subdomain (1.8 ± 1.8), and 1 to 10 on the Written Language subdomain (3.1 ± 3.4). On average, each participant’s Receptive Language score was more than one standard deviation (4.1 ± 3.6) greater than his or her Expressive Language score. Scores on the DP-4 Communication domain ranged from 40 to 77 (57.9 ± 12.7), with the youngest individual having the highest score .disproportionately understand a greater number of words than they could produce. Participants had an average of 13.6 (3.3) of 18 early actions and gestures  and 23.6 (12.4) of 45 later actions and gestures .On the MacArthur–Bates Communicative Indices (MCDI), caregivers reported that participants understood 267.1 (138.9) words out of a total of 396 and produced 56.2 (88.8). On average, participants understood 191 more words than they could produce. While understanding more words than those produced is expected in early stages of development, participants tended to The Beery Visual-Motor Integration, Sixth Edition (VMI-6) was completed by five participants, with standard scores ranging from 42 to 83 (50.8 ± 18.0); four of the five met the criteria for a visual motor integration disorder. The six individuals who could not complete the VMI-6 were not able to independently manipulate a pencil. On the DP-4 Physical domain, participants’ scores ranged from 40 to 68 (47.0 ± 11.1) .On the Developmental Coordination Disorder Questionnaire (DCDQ), participants on average scored 7.8 (2.0) on the Control During Movement domain (maximum 30), 4.1 (0.3) on the Fine Motor/Handwriting domain (maximum 20) and 8.9 (3.8) on the General Coordination domain (maximum 25). Overall, participants scored 20.8 (4.5) on the Total Score; all participants met the threshold for a developmental coordination disorder. On the Vineland-3 Motor domain, participants had standard scores ranging from 52 to 65 (57.8 ± 5.6). Participants showed similar fine and gross motor skills ; Gross MSix of 11 participants presented with inattention and hyperactivity and 5 of 11 with impulsivity. Consensus DSM-5 diagnoses of attention-deficit/hyperactivity disorder (ADHD) were given to 6 of 10 participants . One parEight of 11 participants were reported to present with anxiety. Two individuals presented with obsessive–compulsive disorder (OCD) symptoms—one had a prior diagnosis of OCD. Two presented with aggression and/or self-injury and one with pica. Four participants were receiving medication for mood and/or aggression, including aripiprazole, risperidone, lorazepam, quetiapine, and hydroxyzine.On the Vineland-3, participants had similar levels of internalizing and externalizing behaviors , scoringT-score ranged from 43 to 81 (63.7 ± 12.8), 7 of 11 participants’ scores surpass the clinical cutoff . Six individuals scored above the clinical threshold on the CBCL’s Depressive domain (63.8 ± 10.9), five in the ADHD domain (65.4 ± 9.1), and four in both the Anxiety (63.4 ± 10.9) and Oppositional/Defiant domains (59.2 ± 7.7). On the Aberrant Behavior Checklist (ABC), four individuals scored above the clinical threshold  in the Irritability subdomain, two in the Inappropriate Speech domain, and one in the Hyperactivity domain. Average T scores for the domains were 52.3 (9.6) in Irritability, 52.1 (10.1) in Inappropriate Speech, 50.5 (7.0) in Hyperactivity, 47.5 (7.1) in Lethargy/Social Withdrawal, and 46.9 (6.5) in Stereotypy.The Child Behavior Checklist (CBCL) total The CBCL performed best at predicting ADHD status in our cohort, with five true positives, one false negative, and no false positives . The ABCAgree and Often, respectively.Participants’ caregivers completed the Child and Family Quality of Life (CFQL-2). On average, caregivers rated the Family (3.89 ± 0.98), Caregiver (3.14 ± 0.90), and Child (2.93 ± 0.69) quality of life domains as most affected by their child’s diagnosis of CHAMP1 disorder. Caregivers were less affected in the Financial (2.91 ± 0.84), Relationship (2.86 ± 1.12), and Coping (2.85 ± 0.69) quality of life domains, and they reported that their Social Network quality of life (2.55 ± 0.97) was least affected. The highest rated negative and positive statements were ‘My child’s difficulties have added stress to our home life’ and ‘my child appears happy and content’ where average answers were On the CBCL, caregivers responded to both their greatest concerns about their children and the best things about their children. Six caregivers reported that language ability was one of their top concerns, six reported other DD  as a top concern, and three reported behavioral abnormalities as their top concern. In response to what is best about their children, 10 caregivers described their child as both loving/affectionate and happy/joyful, and 5 caregivers reported that their children are friendly to everyone they meet.n = 4) and hypotonic gait (n = 3). Hyperextensibility was present in 9 of 11 participants. Gastrointestinal abnormalities, present in 9 of 11 individuals, included constipation (8 of 11), gastrointestinal reflux (6 of 11), cyclical vomiting (6 of 11), and diarrhea (1 of 11). Eight participants were treated for these abnormalities: five for constipation, three for reflux, and three for cyclical vomiting.The most common medical comorbidity was hypotonia, which was present in all 11 individuals . CurrentVisual abnormalities were present in 8 of 11 participants, with specific findings of hyperopia (4 of 11), astigmatism (2 of 11), and amblyopia (2 of 11). Ocular abnormalities were also present in 8 of 11, with 5 of 11 having strabismus, 5 of 11 (four current) with nystagmus, and 1 of 11 with a unilateral coloboma.Feeding issues were present in 8 of 11 individuals. Sleep disturbance was present in 7 of 11 and most often included difficulty staying asleep (5 of 11), followed by difficulty falling asleep (3 of 11), restless leg syndrome (1 of 11), and sleep apnea (1 of 11). Four participants were receiving treatment for sleep abnormalities, two receiving melatonin alone, one receiving melatonin and trazodone in combination, and one receiving trazodone and clonidine in combination. Dental abnormalities were also present in 7 of 11 participants, where the most common abnormality was retained primary teeth . Seven of 11 participants had allergies; 5 had seasonal allergies, and 1 of each had mold, food, or penicillin allergies. Six of 11 participants had head abnormalities: 5 of 11 with microcephaly, 2 of 11 with plagiocephaly, and 1 of 11 with macrocephaly.Recurrent infections, defined as two or more severe infections  in one year, three or more respiratory infections in one year, or the need for antibiotics for two months within one year, were present in 4 of 11 participants. Seizures were present in 4 of 11 participants, three with a history of a single febrile seizure and the other with a history of a single febrile seizure and a generalized seizure. One participant had a history of an abnormal electroencephalography, though had never had a seizure. Three of 11 participants required a neonatal intensive care unit (NICU) stay, and 1 of 11 was born prematurely. Additionally, three of nine participants who had undergone an MRI had prominent extra-axial spaces; the remaining seven participants who had an MRI had normal results.Hearing abnormalities were present in 2 of 11 participants, both with bilateral loss. Endocrinology abnormalities were present in 2 of 11 individuals, one with hypothyroidism and one with diabetes mellitus. Features displayed by only one participant include migraines, vesicoureteral reflux, immunodeficiency (as reflected by low T cell counts), and neutropenia.The most common dysmorphic feature was a wide nasal bridge, present in 10 of 11 participants. Eight had a bulbous nose, seven had hypoplastic nails, and six had ear anomalies, which included three with low set ears, two with protruding ears, one with large ears, and one with a preauricular sinus dimple. Five participants had a pointed chin, five had epicanthal folds, four had fleshy hands, four had fifth finger clinodactyly, three had hypertelorism, three had a flat midface, three had a high arched palate, three had two-three toe syndactyly, two had full lips, and two had a short neck. Features present in 1 of 11 include micrognathia, full cheeks, malar hypoplasia, periorbital fullness, deep-set eyes, long eyelashes, and sparse hair. Photos of participants are in This study represents the first prospective comprehensive evaluation of individuals with CHAMP1 disorder. The assessments included multiday clinician-administered evaluations by an interdisciplinary team of clinical researchers. Psychiatric, behavioral, ASD, language, and sensory features were specifically evaluated to fill a gap in the current literature, which has previously focused on medical and dysmorphic features .et al. 2015) or ASD traits  without noting standardized assessment. Consensus DSM-5 diagnoses of ASD were established for three of nine individuals (33%) based on the psychiatric evaluation, the CARS-2obs, and the ADI-R. Results on the ADI-R in this cohort had a high false positive rate (33%), a phenomenon often seen in complex neurodevelopmental conditions due to comorbid ID. However, the sample size is limited. The CARS-2obs, which was used as a remote substitution of the Autism Diagnostic Observation Schedule (ADOS-2), performed marginally better with two false positives. All individuals with a consensus diagnosis of ASD met on both the CARS-2obs and the ADI-R. There was one individual who met on both assessments but did not have ASD—this participant showed clear social strengths, including well-modulated eye contact, initiating interactions, and engaging in social games, and had a range of facial expressions. However, this participant was difficult to engage in more structured remote assessment, which likely attributed to the false positive on the CARS-2obs. Additionally, regardless of DSM-5 ASD diagnosis, many individuals in our cohort presented with features of ASD. Most individuals, regardless of ASD status, presented with RRBs and sensory symptoms, a finding seen in other genetic NDDs  and OCD traits (18%). Half of those diagnosed with ADHD were receiving pharmacological interventions. We found that the CBCL performed best at assessing ADHD features in our cohort, as compared to the ABC Hyperactivity domain. There were also high rates of anxiety reported in this cohort (73%), with three participants having separation anxiety. Despite this, in line with previous reports, many individuals presented with a happy and joyful demeanor . This waWe observed wide variability in cognitive and adaptive functioning, ranging from severely impaired to scores within the low average range. One explanation for the variability is the wide age range within our cohort (1.6–28.3 years). Domain scores on the DP-4 and Vineland-3 were significantly negatively associated with age, an expected finding. The youngest participant in our cohort had the highest scores in all domains on both assessments, and the oldest participant had the lowest scores. Because expectations for developmental and adaptive functioning skills are lower in younger children, standard scores—which compare current skills to those expected for age—tend to be higher. As the breadth of skills expected increases with age, the margin of deficits in these individuals becomes wider, and scores therefore commonly decrease with age. In addition to age, we found that microcephaly was associated with lower scores on the Vineland-3 Adaptive Behavior Composite, Communication, and Daily Living Skills domains.Previous literature has noted differing rates of verbal ability; combining all studies to date, 67% of assessed individuals with CHAMP1 disorder attained verbal language, at minimum at the single-word level. Results from clinician-administered direct assessments, caregiver interviews, and caregiver questionnaires indicated that most individuals in our cohort had higher receptive vocabulary and language ability compared to expressive. Most of our cohort utilized single words (9 of 11) while only four used spontaneous phrase speech. However, three participants were still younger than this cohort’s average age of phrase speech acquisition at the time of assessment and may achieve this milestone later on. There was no report of language regression.ADNP and CHAMP1 interact with similar genes and pathways, including HP1. While not the same abnormalities, given the biological connection between the genes, it is interesting that both conditions have primary tooth abnormalities and may hint at shared pathways.Medical features are the most thoroughly described comorbidities in previous literature. Our study found similar rates of common features such as microcephaly, hypotonia, visual abnormalities, dysmorphic features, and gastrointestinal abnormalities. Gait abnormalities were prospectively assessed for the first time and found to be universally present in this cohort (10 of 10). Additionally, endocrine abnormalities  were present in 2 of 11 participants, a new finding to the literature. Interestingly, four of seven individuals had retained primary teeth. In another NDD, ADNP syndrome, the majority of individuals have premature primary tooth eruption . ADNP anThe battery of assessments utilized in this study was designed to be conducted fully remotely due to safety concerns of in-person testing during the COVID-19 pandemic. However, beyond the specific need for a virtual protocol, we found concrete benefits of conducting the study remotely. First, clinicians were able to observe and assess participants in their homes. Typically, families travel to our center for multiday evaluations, and the change in schedule and environment can impact behavior. Assessing individuals in their natural environments may mitigate these difficulties and lead to more accurate assessments of behavior that are representative of everyday life. Second, while socioeconomic status and geographic location are typically barriers to research participation, we were able to increase accessibility in this study. The remote battery eliminated the need for families to take off multiple days of work and the financial burden of having to travel to complete the study.However, there were also limitations to the remote battery, including the inability to complete both traditional cognitive testing and the ADOS-2. While ASD consensus diagnoses were given with high certainty in most cases, we deferred one diagnosis because of insufficient evidence with the available format. The addition of the ADOS-2 could have helped to clarify ASD status in this participant. Additionally, there was one participant who did not complete the full battery because of loss to follow-up, which may have been prevented with in-person visits. Overall, we found the benefits of the remote study outweighed the limitations. In the future, we will likely utilize a combination of remote and in-person assessments to reduce the burden to families while also comprehensively collecting data. Co-norming this battery of assessments is also an important future direction and is necessary to optimally examine which domains are most versus least impaired. Co-norming will offer a useful metric at both the group and individual level, particularly in syndromes such as CHAMP1 that often result in impairment across multiple domains of functioning.CHAMP1 therefore warrants inclusion on ASD-focused sequencing panels. Sensory reactivity symptoms were also notably common and suggest the importance of thorough assessments of the sensory domain and the potential utility of related interventions such as occupational therapy. Future studies can also investigate whether sensory symptoms are affecting other core domains such as social and behavioral features. High rates of ADHD (60%) but low rates of consistent treatment (50% of those with ADHD) indicate that treatment for ADHD is underutilized and provides another promising target of therapeutic intervention for individuals with CHAMP1 disorder. Additionally, a review of clinical measure performance in this cohort may aid in clinical trial design to determine optimal endpoints and clinical outcome assessments. Importantly, this study provides further characterization of sensory, language, motor and medical features and serves as a basis for the feasibility of future remote phenotyping studies in rare neurogenetic syndrome.Overall, this study provides a comprehensive neurobehavioral profile of individuals with CHAMP1 disorder. While rates of ASD are relatively low in this rare disorder compared to others such as tuberous sclerosis or ADNP syndrome, they are higher than some other disorders already included on clinical autism sequencing panels (e.g. FOXP1). Though the sample size was small, our results provide evidence that ASD is a common comorbidity in CHAMP1 disorder, and CHAMP1 gene as classified by the American College of Medical Genetics and Genomics and Association for Molecular Pathology guidelines (Participants ranged from 1.6 to 28.3 years of age (10.7 ± 7.8) and included eight females and three males. All participants had a diagnosis of CHAMP1-related NDD, confirmed by a likely pathogenic or pathogenic sequence variant in the idelines .Clinical assessments included approximately four to six hours of direct evaluation of the participant, six hours of caregiver interviews, and four hours of caregiver questionnaires . All direct evaluations and caregiver interviews were conducted remotely using videoconferencing (HIPAA-compliant Zoom). Questionnaires were administered virtually using an online system, REDCap. Assessments were administered by a child and adolescent psychiatrist, clinical psychologists, a pediatric neurologist, and a genetic counselor. Additionally, medical records were reviewed by the study psychiatrist to supplement medical history information.Developmental milestones were collected using the ADI-R , the EarThe Developmental Profile 4  was usedobs  or by intellectual quotient under 70 (n = 1), per the manual (Psychiatric comorbidities were evaluated during the psychiatric evaluation and using the ABC  and the e manual . ConsensThe CFQL-2  was usedMedical comorbidities were assessed during the psychiatric evaluation and supplemented with the review of prior medical records by the study psychiatrist. The neurological evaluation assessed gait, tone and motor coordination through remote assessment and by review of videos collected for this purpose. Dysmorphisms were assessed by a genetic counselor through videoconferencing and caregiver interview.Supplementary_Tables_1-4_ddac018Click here for additional data file.


Objectives. To identify occult urolithiasis and nephrocalcinosis in asymptomatic patients with NPHPT and evaluate biochemical markers as risk predictors for the development of renal calcification (RC). Methods. Cross-sectional analysis of 34 patients with no history of urolithiasis and/or nephrocalcinosis. The diagnosis of NPHPT was as follows: elevated serum PTH (reference range: 15–65 pg/mL), normal albumin-corrected serum calcium, normal urinary calcium excretion, serum 25(OH)D >30 ng/mL, eGFR (CKD-EPI) > 60 mL/min/1.73 m2, without intestinal disease, and not on medications such as thiazide diuretics, lithium, bisphosphonates, or denosumab. Patients were categorized according to the presence or absence of RC identified by renal imaging. Their clinical and biochemical characteristics were then compared. Results. The patients had a mean age of 67.97 ± 10.45 years, predominantly postmenopausal women (88.2%); serum PTH, 119.67 ± 64.44 pg/mL; 25(OH)D, 39.00 ± 8.88 ng/dL; 1.25(OH))2D, 74.53 ± 26.37 pg/mL; corrected serum calcium, 9.34 ± 0.62 mg/dL; and 24-hour urinary calcium, 134.87 ± 79.68 mg/day. RC was identified in 26.5% of the patients. There was no difference in anthropometric and clinical parameters, renal function, 25(OH)D, and urinary pH in patients with or without RC. Patients with RC had higher PTH values , 1.25(OH) 2D , and 24-hour urinary calcium . Conclusion. Occult renal calcifications are common in NPHPT and are associated with increased serum PTH, 1.25(OH))2D, and 24 h urinary calcium.Normocalcemic primary hyperparathyroidism (NPHPT) is characterized by elevated serum levels of parathyroid hormone (PTH) with persistently normal serum calcium concentrations after excluding secondary causes of hyperparathyroidism. Urolithiasis and/or nephrocalcinosis may occur in hypercalcemic PHPT, but little is known about these complications in NPHPT.  Secosurgery) –6.In the last decade, an increase in the prevalence of NPHPT has been observed, in addition to the recognition of clinical complications that were previously found in the classic and hypercalcemic forms of primary hyperparathyroidism (PHPT) –10. The This study aims to investigate occult renal calcifications in patients with NPHPT and their associated risk factors.Forty-five patients were selected from our endocrine outpatient clinic. Eleven patients with a history of urolithiasis or nephrocalcinosis were excluded. The diagnosis of NPHPT was based on the following criteria: serum PTH above the reference range  and serum calcium within the normal range (8.4–10.4 mg/dL), with values measured simultaneously and confirmed on at least 2 occasions, normal urinary calcium excretion (<4 mg/kg/24 h), serum 25(OH)D >30 ng/mL, eGFR >60 mL/min/1.73 m2, without intestinal diseases, and not on medications such as thiazide diuretics, lithium, bisphosphonates, or denosumab.2D, albumin, phosphorus, urea, and creatinine, in addition to 24-hour urine calcium and urinary pH. Measurement of PTH and 25(OH)D was performed by electrochemiluminescence , and 24-hour urinary calcium was assessed by calorimetry. Serum 1.25(OH)2D was measured by liquid chromatography tandem mass spectrometry. Albumin-adjusted calcium was used for this study and calculated according to the formula proposed by Figge et al. [Patients who agreed to participate, after signing an informed consent form, answered a specific questionnaire and underwent a complete physical examination. Blood was collected after an overnight fasting for laboratory tests including PTH, 25(OH)D, total calcium, 1.25(OH)e et al. :(1)Adjus2D is 18–72 pg/mL.The reference range for adjusted calcium is 2.10–2.55 nmol/L (8.4–10.4 mg/dL), for PTH is 15–65 pg/mL, and for 1.25(OH)The eGFR was calculated using the chronic kidney disease epidemiology collaboration (CKD-EPI) equation:The patients underwent imaging studies for evaluation of renal calcification by computed tomography (CT)  or ultrasound (US) with 3–7 MHz transducers . Half of the patients underwent CT imaging.Patients were categorized into two groups based on the presence or absence of renal calcification, according to the result by the imaging method.Microsoft Office Excel was used for data collection and the IBM-SPSS version 25 program for statistical analysis. Descriptive statistics were expressed as absolute (N) and relative (%) frequency for categorical variables. For continuous variables, mean and standard deviation (SD) were used to describe variables with normal distribution and median and interquartile range for nonnormal distributions.t-test with equal variances for independent samples or the Mann–Whitney test was used. To determine the discriminatory power of the presence of kidney stones, a ROC curve was used for each variable, PTH, 1.25(OH)2D, and 24 h urinary calcium (mg/24 h), to calculate the area under the curve, test, and confidence interval for measure, sensitivity values, and specificity for the value that maximizes the sum of the sensitivity and specificity measures. The significance level used in interpreting the statistical test was 5%.To compare the two groups  in relation to numerical variables, Student's 2. Laboratory results were as follows (mean ± SD) or median (interquartile range): serum PTH 99.80 (83.83–131.7) pg/mL, 25(OH)D 39.00 ± 8.88 ng/mL, 1.25(OH)2D 74.53 ± 26.37 pg/mL, albumin-corrected serum calcium 9.34 ± 0.62 mg/dL, phosphorus 3.45 ± 0.57 mg/dL, urinary pH 6.43 ± 0.50, and 24 h urinary calcium 127.50 (70.25–188.00) mg/day , with mean age of 67.97 ± 10.45 years and BMI of 26.17 ± 3.57 kg/m) mg/day .Of the 34 patients, 9 (26.5%) had occult urolithiasis or nephrocalcinosis identified by CT or US. Two patients had nephrocalcinosis (5.9%) and 7 patients had urolithiasis (20.6%), with microcalculi size ranging from 1 to 9 mm. Among patients with urolithiasis, stones were identified as follows: 2 patients with a unilateral microcalculi (6 and 9 mm), 3 patients with bilateral microcalculi ranging from 5 to 7 mm, 1 patient with 3 unilateral microcalculi ranging from 1 to 2 mm, and 1 patient with 4 unilateral microcalculi ranging from 1 to 5 mm. All patients underwent investigation of renal calcification with US of the urinary tract, with 50% of the sample performing complementary CT. Renal calcifications were identified in 20.6% of patients who underwent US and in approximately 30% of those who underwent CT.P = 0.001), 1.25(OH)2D , and urinary calcium  and lower values for serum phosphate   . There w2D, and 24-hour urinary calcium had an area under the curve (AUC) of 0.850 , 0.894 , and 0.731 , respectively. Serum PTH levels >101.30 pg/mL  and 1.25(OH)2D > 69.50  were identified in all patients with renal calcifications. Twenty-four hour urinary calcium was also associated with the presence of occult calcification from 137 mg/day with sensitivity and specificity greater than 70% than 70% .Renal calcification, which includes urolithiasis and nephrocalcinosis, is the most common complication of hypercalcemic PHPT , 13, butWith regard to the general population, urolithiasis has a lifetime prevalence of approximately 11% in men and 7% in women, according to data from the National Health and Nutrition Examination Survey (NHANES) . In EuroThe high rate of urolithiasis and nephrocalcinosis, even in asymptomatic patients with NPHPT, indicates that complications associated with PHPT can be an early event , and thiIn this regard, Ejlsmark-Svensson et al. , using CThe similarity in the prevalence of urolithiasis between normocalcemic and hypercalcemic patients has been retrospectively reported by our group suggesting that NPHPT may not be a behaviour as an indolent condition .P = 0.008) [In a recent study with 96 asymptomatic patients with hypercalcemic PHPT, a high incidence of occult urolithiasis (21%) was observed when active searching by renal imaging was done . The hig= 0.008) . In the = 0.008) .US has lesser sensitivity (40% versus 97%) and specificity (84% versus 96–100%) when compared with CT for diagnosing renal stones, especially in the detection of stones smaller than 3 mm –28. By i2D, and urinary calcium were found in patients with renal calcifications compared to those without calcifications, and these parameters can be predictors of urolithiasis/nephrocalcinosis. This is in agreement with other studies in hypercalcemic PHPT [In the present study, higher serum PTH, 1.25(OH)mic PHPT , 25.2D was not measured in this study.Of note, using US only, another study from our group was unable to show any differences in serum PTH or urinary calcium between NPHPT patients with or without kidney stones . Althoug2D synthesis through renal hydroxylation of 25(OH)D, which, in turn, may increase filtered calcium load and predispose to hypercalciuria and renal calcifications [2D may influence calcium sensing receptor (CaSR) regulation and calcium channel expression, this may well be also a contributing factor for kidney stone formation as it would lead to a lower calcium resorption in the kidney and thus higher urinary calcium [High serum PTH concentrations would promote greater stimulation of 1.25(OH)ications , even wi calcium , 31.Other factor that must be taken into account is some degree of PTH resistance in the kidney seen in patients with PHPT , 32. SciThe present study has some limitations. The cross-sectional design, the lack of a control group and ionized calcium measurements, although in at least two occasions, serum albumin-corrected serum calcium were below 10.3 mg/dL in all patients. 25(OH)D levels below 30 ng/mL were adopted as exclusion criteria, although some authors establish a threshold of 40 ng/mL for the diagnosis of NPHPT. Also, we do not have data regarding the patient's diet and urinary sodium and parameters that could influence the risk of hypercalciuria and renal calcifications. As strength, we were able to identify risk factors related to kidney calcifications in NPHPT, an area where research is urgently needed.2D, and 24 h urinary calcium.In conclusion, our data showed that occult renal calcifications are common in NPHPT and are associated with increased serum PTH, 1.25(OH)


Tpref) of individual zebrafish (Danio rerio). AS was greatest 2.5°C above Tpref, which was driven by an increase in maximal metabolic rate. This mismatch between Tpref and the optimal temperature for AS suggests that factor(s) aside from AS maximisation influence the thermal preference of zebrafish.Warming is predicted to have negative consequences for fishes by causing a mismatch between oxygen demand and supply, and a consequent reduction in aerobic scope (AS) and performance. This oxygen and capacity limited thermal tolerance (OCLTT) hypothesis features prominently in the literature but remains controversial. Within the OCLTT framework, we hypothesised that fish would select temperatures that maximise their AS, and thus their performance. We tested this hypothesis using intermittent flow respirometry to measure AS at, above (+2.5°C) and below (–2.5°C) the self-selected, preferred temperature ( Summary: Individual zebrafish select temperatures below their optimum for aerobic scope. Global temperatures are increasing and will continue rising until at least 2050 . Rising TOptAS) pushes an animal to its pejus temperatures, resulting in a reduction in physiological performance and biological fitness , where loss of equilibrium or ‘ecological death’ occurs. Given the central importance of AS within the OCLTT framework, we hypothesised that fish would select the environmental temperature that produces the greatest AS at a given time. This hypothesis has previously been tested in other studies: juvenile barramundi  behaviourally select temperatures ∼6°C lower than those that maximise AS (Petromyzon marinus) Tpref and TOptAS roughly align  select a temperature based on AS maximisation by measuring AS at, above, and below the temperature preference (Tpref) of individuals.One temperature-dependent trait that has received a lot of interest from the scientific community over the past two decades is aerobic scope (AS) . AS is t fitness . The furimise AS , whilst Danio rerio AB strain, mass=0.62±0.019 g (mean±s.e.m.), mixed sex, aged 12–18 months] were raised and maintained at The University of Manchester, UK. Fish were held at 28°C in a 14 h light:10 h dark cycle prior to the experiments. Fish were fasted for 12 h prior to experimentation to achieve a post-absorptive state . Two protocols  of 13 zebrafish was determined individually using a custom-built shuttlebox system  or cold (4°C) reservoirs through titanium plates under the base of the experimental chambers. A stick heater was present in each chamber and, when required, was manually used to maintain the 2°C difference between chambers if any drift occurred. A re-circulating pump in each chamber mitigated thermal stratification and was positioned to limit mixing between the warm and cold chambers. Temperature in each chamber was measured every 30 s using an Elitech RC-4 temperature data logger (±0.05°C)   . The finn period . After tN=12) used in the Tpref trials. Each fish had one trial at its Tpref, one 2.5°C above its Tpref and one 2.5°C below its Tpref [Tpref=28.63±0.53°C (mean±s.e.m.); range, 25.55–31.42°C; N=12; −1 prior to AS measurement, which occurred as soon as the test temperature was reached. Trial order was randomised, and a minimum of 24 h left between trials to allow for recovery. The trial order for individual fish was determined using a random number generator.Respirometry trials were performed to assess maximal metabolic rate (MMR) and standard metabolic rate (SMR) to calculate AS. For protocol A, three respirometry trials were performed on the individual zebrafish  to re-oxygenate the system and prevent the waste products of metabolism accumulating. In the wait period, the chamber was functionally sealed so the oxygen content of the water declined linearly based on the rate at which the fish extracted oxygen. Finally, during the measurement phase, the linear decline in oxygen content over time was measured and used to calculate the fish's aerobic metabolic rate. AquaResp 3.0 was used for the automation of aquatic respirometry .Oxygen uptake rate was measured using a custom-built intermittent flow respirometry system. Four 100 ml (including the volume of associated tubing) Plexiglass chambers were submerged in a 60 l header tank that was supplied with temperature controlled (±0.3°C), aerated, filtered (200 µm filter) water from a 40 l buffer tank. Water was mixed within the chambers through a re-circulating loop where oxygen content was measured every second . Each respirometry loop consisted of a 60 s flush phase, 120 s wait period and 300 s measurement phase. During the flushing phase, water from the buffer tank was pumped into the chambers  prior to being transferred through the air (standardised to 30 s) and placed in the respirometry chamber during the wait phase. Chasing typically occurred in the afternoon, following which the fish were left in the dark until the following morning to reach standard metabolic rate (SMR). Owing to the relatively long wait and measurement phases (420 s combined), MMR is likely to be underestimated in our study; however, this underestimation is probably consistent between our groups and accordingly should not influence our conclusions. Prior to, and immediately following each experiment, background respiration in the system was measured (over 1500 s). Following each experiment, the fish were returned to their holding tank and the respirometry system was bleached, drained, rinsed and re-filled for subsequent trials.https://www.r-project.org/) was used to process the respirometry data. MMR was calculated as the steepest slope (r2 ≥0.95) recorded in the first hour of recordings following the chase. SMR was calculated as the lowest 10% of values (r2≥0.90) recorded throughout the remaining portion of the trial. MMR and SMR were background corrected using the linear relationship between background respiration and time to subtract an appropriately weighted background respiration measurement from each oxygen uptake measurement. Background respiration (mean±s.e.m) accounted for 18.6±0.91% and 40.8±2.19% of uncorrected MMR and SMR, respectively. AS was calculated as MMR–SMR using the background respiration corrected data.FishResp v.1.0.4  in R v.4r2=0.87, MMR; r2=0.93) and Gaussian  functions, respectively. AS, SMR and MMR were compared across temperatures using one-way ANOVAs in Prism.The relationships between mean SMR, MMR and AS against temperature were modelled in GraphPad Prism v.9.1.0 with second order polynomial  and lmerTest (https://CRAN.R-project.org/package=lmerTest) mixed effects models in R v.4.1.2 (Tpref) and trial order were included as fixed effects, and individual included as a random effect. The residuals from each model conformed to normal distributions , and pairwise comparisons between temperature groups were computed using emmeans (https://CRAN.R-project.org/package=emmeans) in R v.4.1.2.SMR, MMR and AS when measured 2.5°C above, 2.5°C below and at each individual's  v.4.1.2 . TemperaTpref =28.63±0.53°C (mean±s.e.m.), range 25.55–31.42°C, N=12; TOptAS) , but there was no change in SMR  are known to be misaligned (TOpt for AS and escape velocity in spiny lobster (Sagmariasus verreauxi) larvae, but not juveniles  hypothesis emphasises a key role for AS in the physiology of aquatic ectotherms . OCLTT psaligned , as are uveniles . Given tTOptAS, implying that traits other than AS dictate the preferred, and thus likely optimal, temperature of zebrafish. Previous research supports this conclusion, with a study on juvenile barramundi  measuring Tpref as 31.7°C, but showing an increase in AS up to 38°C (The multiple performances multiple optima (MPMO) hypothesis, like OCLTT, recognises that AS is a temperature-dependent trait that contributes to an organism's biological fitness . However to 38°C .Tpref in individual zebrafish, at the ‘population level’ (here defined as different groups of fish used at each temperature), there was no significant relationship between AS and temperature  owing to increasing MMR and SMR  with similar Q10 values . Superficially, this lack of relationship between AS and temperature appears to contradict the observation that AS is more than 2.5°C above Tpref rather than at Tpref. However, considering the intraspecific variation in Tpref measured in this study , temperatures that were close to Tpref for some individuals would be below, or above, the Tpref of others. This variation in Tpref and likely TOptAS may preclude any population-level relationship between temperature and AS because, across any given temperature change, the AS of individuals within the population would be changing in different ways. Warming waters may therefore be favouring certain individuals within a population, even when population-level effects of warming are not apparent. Zebrafish have a broad TOptAS (TOptAS (Despite AS increasing at 2.5°C above d TOptAS B, as is  (TOptAS , show thOceans and rivers are warming at an unprecedented rate. A recent IPCC report suggests that under all probable emissions scenarios, global surface temperatures will continue to rise until at least the middle of the 21st century, with temperature increases expected to exceed 1.5°C . As wate10.1242/jexbio.243774_sup1Supplementary informationClick here for additional data file.


Paenarthrobacter ureafaciens. The P. ureafaciens ZY demonstrated a significant degradation capacity of atrazine, with the degradation efficiency of 12.5 mg L−1 h−1 in liquid media . The P. ureafaciens ZY contained three atrazine-degrading genes  could metabolize atrazine to form cyanuric acid, which showed lower biotoxicity than the parent atrazine as predicted by Ecological Structure Activity Relationships model. A laboratory-scale pot experiment was performed to examine the degradation of atrazine by P. ureafaciens ZY inoculation and investigate its effects on the native microbial communities. The results exhibited that the P. ureafaciens ZY was conductive to the degradation of atrazine, increased the total soil phospholipid fatty acids at the atrazine level of 50, 70, and 100 mg kg−1. By using high-throughput sequencing analysis, Frateuria, Dyella, Burkholderia-Caballeronia-Paraburkholderia were considered as the most important indigenous atrazine-degrading microorganisms due to their relative abundances were positively correlated with the atrazine degradation rate. In addition, P. ureafaciens ZY also increased the abundance of atrazine-degrading genus Streptomyces and Bacillus, indicating that there may be a synergic relationship between them in the process of atrazine degradation. Our work provides a new insight between inoculums and native microorganisms on the degradation of atrazine.An atrazine-utilizing bacterium, designated as ZY, was isolated from agricultural soil and identified as  In fact, as little as 1% of applied pesticides reaches the target weeds and pests, and a more significant proportion of pesticides residues in the soils will lead to toxic effects on the ecological system and human health , a synthetic herbicide, has been extensively applied for more than 50 years. Its application has been controversial due to its widespread, high fluidity, and persistence, resulting in its high residues in soils . However elegans , Chirono tentans , Daphniacarinata , Elodea nadensis , Lepomisrochirus , Litodits marina , Pimephapromelas , Prochillineatus , and so Achromobacter  after inoculation for 20 days, respectively. A 96.86% degradation percentage of atrazine was observed with Rhodobacter sphaeroides sp. W16 inoculation in the soil at the level of 100 mg kg−1 atrazine after 15 days  that affect atrazine degradation were explored. The genes and metabolic pathways involved in atrazine degradation by strain P. ureafaciens ZY were elucidated. Besides, this work also investigated the degradation capacity of P. ureafaciens ZY in the soil and its effects on the dynamic of soil native microbiome through PLFAs and high-throughput sequencing analysis. Our work enriched the species of bacteria that could degrade atrazine and provided a theoretical basis for the remediation of atrazine-contaminated soils.In this study, a strain with a high-efficiency degradation capacity of atrazine, 2HPO4, 0.2 g MgSO4·7H2O, 1.2 g KH2PO4, 0.025 g CaCl2·2H2O, 0.008 g Fe2 (SO4)3, and 1.0 g glucose in 1,000 ml of deionized water at pH 7.0 (marked as MSMG in this study). The liquid LB media contained 10 g tryptone, 5 g yeast extract, and 10 g NaCl in 1,000 ml of deionized water at pH 7.0. Solid media was formed by adding 17 g L−1 agar into liquid media, and all media were sterilized at 121°C for 30 min for further used.Soil samples were collected from agricultural soil in the northeast of China  and were sieved to remove plant residues and stones for further study . Atrazine was purchased from Shanghai Macklin Biochemical Co., Ltd. Cyanuric acid was purchased from Heowns Biochem Technologies, LLC, Tianjin. The liquid minimal salt media (MSM) amended with glucose as a carbon source was consisted of 2.4 g K−1 atrazine) and incubated under anaerobic conditions at 30°C and shaken at 160 rpm for 7 days. Then, the enrichment culture (10 ml) was transferred into a fresh MSMG (contained 200 mg L−1 atrazine) and incubated for 7 days at the same conditions. The inoculation process was repeated five times until the final atrazine concentration reached 500 mg L−1. Subsequently, the culture was collected and plated onto MSMG agar plates contained 100 mg L−1 of atrazine and incubated at 30°C in the darkness. After 4 days of incubation, single colonies were selected aseptically and explored their degradation ability for atrazine. A strain designated as ZY with atrazine degradation activity was obtained for further analysis.Soil samples (10 g) were added to 100 ml of MSMG  was carried out to test carbon substrates utilization patterns of strain ZY by using automated tetrazolium-based microbial identification system produced by BIOLOG Inc.  was used to extract the total genomic DNA of strain ZY. Strain ZY was identified 8 CFU mL−1 (OD600nm = 1.0) determined by a UV-spectrophotometer  was obtained for subsequent degradation experiments.The strain ZY was cultured overnight (18 h) in LB media at 30°C and shaken at 160 rpm. After centrifugation, the strain cells were collected and suspended three times in MSMG media. A final cells density of 1 × 10−1. Only one parameter was changed during each culture. The MSMG suspension with OD600nm 1.0 were added into the fresh MSMG media at 1:50 (v:v) dosage of inoculation and cultured at 30°C. The OD600nm and atrazine in the culture were detected every 2 h. Atrazine and cyanuric acid were determined by ultra-performance liquid chromatography (UPLC) analysis . The primer pairs used in this study were described in via BLAST on NCBI databases.The strain ZY was analyzed by PCR for the presence of atrazine-degrading genes  technique. Specific UPLC-Q-TOF-MS experimental parameters and the mass spectrometry methods were performed as described by previous studies (To identify the atrazine metabolites produced by  studies .−1 (dry weight) and then was placed in a fume hood for 4 h to volatilize methanol. The soils were inoculated with strain ZY to reach the initial concentration of 4 × 106 CFU g−1, then passed through a mesh (2 mm) to achieve symmetrical. The treatments with ZY inoculums at 0, 10, 30, 50, 70, and 100 mg kg−1 atrazine were marked as P0, P10, P30, P50, P70 and P100, respectively. Soil samples containing atrazine at 0, 10, 30, 50, 70, and 100 mg kg−1 but without ZY inoculums were labeled as A0, A10, A30, A50, A70, A100, respectively. Then, soil samples were transferred to a plastic bowl (Φ 9 cm) and incubated in darkness at 25°C with 30% water-holding content adjusted by sterile distilled water. After inoculation for 1, 3, 5, and 7 d, soil samples were collected to analyze residual atrazine. In brief, 3 g of soil samples were added with 15 ml methanol for ultrasonic for 20 min, followed by centrifugation for supernatant collection, and the above operations were repeated three times (The 200 g soil sample (dry weight) was added with methanol-dissolved atrazine to reach final concentrations of 10, 30, 50, 70, and100 mg kgee times . The recThe PLFAs analysis was used to assess soil microbial community structures. The PLFAs were extracted and analyzed using the methods reported by The Illumina MiSeq of PCR-amplified 16S rRNA was used to analysis the structure of soil microbial community. Total DNA of soil was extracted by using the Cetyltrimethyl Ammonium Bromide (CTAB) according to manufacturer’s instructions. The 16S rRNA gene fragment was amplified using a universal primer set 341F (5’-CCTACGGGNGGCWGCAG-3′) and 805R  model was used to predict the toxicity of atrazine metabolites. Principal component analysis (PCA) was performed by CANOCO 5.0 software. Heat map analysis and network analysis were performed using the R package (v3.5.2).A strain ZY with a strong atrazine-degrading ability was selected for further study. A light yellow, smooth, and round colony of strain ZY was observed in the LB plate . Its morP. ureafaciens NC , P. nicotinovorans DSM 420 , and P. histidinolovorans DSM 20115 . In a phylogenetic tree constructed based on the similar 16S rRNA gene sequences of ZY , very similar to those of accessible strains, e.g., es of ZY , the strP. ureafaciens ZY was investigated via GEN III microplate after 48 h of incubation. The results have shown in P. ureafaciens ZY can metabolize D-fructose, D-mannose, D-galactose as carbon sources, but cannot assimilate dserine, troleandomycin, rifamycin, and minocycline  at different pH levels for 6 h. After inoculation for 6 h, more than 64.83% of atrazine removal were observed at pH 5.0–11.0. At pH 3.0, P. ureafaciens ZY hardly grew and could not metabolize atrazine. The degradation rates of atrazine were 11.42, 12.11, 11.21, and 10.81 mg L−1 h−1 at pH ranging from 5 to 11, respectively. The pH range for the growth and atrazine degradation of P. ureafaciens ZY is wide and alkaliphilic, which also revealed that P. ureafaciens ZY had a great potential to rete atrazine-contaminated sites, particularly in alkaline sites. In addition, there was a significant correlation between the atrazine degradation percentage and OD600nm from pH 3 to 11 , suggesting that pH was an important factor affecting strain proliferation and atrazine degradation.Environmental pH could influence the microbial remediation of contaminated sites . The opt5.0–10.0 . Figure −1 atrazine in MSMG (pH 7.0). With the increase of temperature, the degradation of atrazine enhanced and degradation rates were 8.08, 8.20, and 11.95 mg L−1 h−1 at 20, 25, and 30°C after 6 h, respectively. Notably, the degradation ability of atrazine were inhibited at 35°C and 40°C after 6 h. Citricoccus sp. strain TT3 was 30°C, and the growth and atrazine degradation of Citricoccus sp. strain TT3 were inhibited at 37°C and 45°C. In addition, a significant correlation between the atrazine degradation percentage and OD600nm from 20 to 40°C  was observed, indicating that strain proliferation and atrazine degradation could be affected by the temperature.Temperature has played a key role in biodegradation efficiency of pollutants by microorganisms, and significantly influences the dissipation of s-triazine herbicides . Figure −1 on the growth and atrazine degradation of P. ureafaciens ZY were explored in MSMG at pH 7.0 and 30°C , the growth of P. ureafaciens ZY was obviously promoted , atzB, and atzC, which could catalyze atrazine to produce cyanuric acid (atzD (trzD), atzE, and atzF to mineralize atrazine into CO2 and NH3  exhibited 100% identity with the trzN gene of Arthrobacter sp. AK-YN10 (HE716868.1) and Pseudomonas sp. ADP (FJ161692.1). The atzB  showed 100% homologous with the atzB gene of Arthrobacter sp. ZXY-2 (CP017421.1) and P. ureafaciens DnL1-1 (CP014574.1). The atzC  showed 99.67 and 99.67% homologous with the atzC gene of Pseudomonas sp. ADP (U66917.2) and Nocardioides sp. SP12 (AF537329.1). During the degradation process of atrazine by P. ureafaciens ZY, three metabolites of atrazine, were identified as hydroxyatrazine (m/z of 198.0), N-isopropylammelide (m/z of 171.0), and cyanuric acid (m/z of 128.0) by UPLC-Q-TOF-MS  to form cyanuric acid. Further determination of cyanuric acid content can also confirm the degradation path of atrazine  of atrazine compared to parent atrazine  was found in A30 after 7 days. In the treatments of A10, A50, A70, and A100, the degradation of atrazine was 12.71%, 12.87%, 13.03%, and 7.44%, respectively . These results indicated that P. ureafaciens had great potential for remediation of atrazine-contaminated soil.The biodegradation of different concentrations of atrazine was further investigated in soil with or without ectively . Atrazinectively . In receP. ureafaciens ZY showed a high degradation rate for atrazine in soil. In this work, the freshly added soils were used, and the addition of −1) is higher than those in other treatments, especially in A70  and A100 . Among them, the Gram-positive , Arthrobacter/Citricoccus (Actinobacteria), Bacillus (Firmicutes), respectively , 1.11% (P10), 2.56% (P30), 2.75% (P50), 4.39% (P70) and 3.30% (P100) at 7 days, respectively. These results showed that the inoculated P. ureafaciens ZY could survive and proliferate in atrazine-contaminated soils. In our study, the atrazine degradation rate was positively correlated with Frateuria (p < 0.01), Dyella (p < 0.05), Burkholderia-Caballeronia-Paraburkholderia (p < 0.05), and Paenarthrobacter (p < 0.01), respectively. Compared to A0, an increase of relative abundance of 16.71%–312.53% (Frateuria) and 7.09%–66.93% (Dyella) were found in other groups. Atrazine alone addition decreased the relative abundance by 1.49%–26.73% in Burkholderia-Caballeronia-Paraburkholderia, however, P. ureafaciens ZY increased the relative abundance of Burkholderia-Caballeronia-Paraburkholderia by 31.68%–221.78% compared to those in non-inoculation treatments. The previous studies had reported that Frateuria species could metabolize amine . In addition, Frateuria, Dyella, and Burkholderia-Caballeronia-Paraburkholderia showed significantly positive correlations with atrazine degradation rate, suggesting that they were also important participants in the atrazine degrading process. Moreover, there may be a synergic relationship between P. ureafaciens ZY, Streptomyces and Bacillus during atrazine degradation. Our work provides a theoretical basis for bioaugmentation in atrazine-contaminated soil.This study isolated and identified a novel atrazine degrader The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number (s) can be found in the article/YZ: conceptualization, investigation, data curation, Writing—original draft. XL: supervision, funding acquisition, writing—review. YL: methodology, writing. HB: methodology, writing. JN: supervision, Writing—review and editing. GX: Writing—review and editing. All authors contributed to the article and approved the submitted version.National Key Research and Development Program of China (2019YFC1906501 and 2018YFC1901100).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Pseudoalteromonas sp. Xi13 can degrade κ-selenocarrageenan to selenium oligosaccharides. The maximum optimized κ-selenocarrageenase activity using Response Surface Methodology (RSM) was increased by 1.4 times, reaching 8.416 U/mL. To expand applications of the κ-selenocarrageenase in industry, the preparation conditions of it in either lyophilized or immobilized form were investigated. The activity recovery rate of the lyophilized enzyme was >70%, while that of the immobilized enzyme was 62.83%. However, the immobilized κ-selenocarrageenase exhibits good stability after being reused four times, with 58.28% of residual activity. The selenium content of κ-selenocarrageenan oligosaccharides degraded by the immobilized κ-selenocarrageenase was 47.06 µg/g, 8.3% higher than that degraded by the lyophilized enzyme. The results indicate that the immobilized κ-selenocarrageenase is suitable for industrial applications and has commercial potential.The bioenzymatic production of selenium oligosaccharides addresses the problems resulting from high molecular weight and poor water solubility of κ-selenocarrageenan, and lays foundation for its application as adjuvant drugs for cancer treatment and food additive. κ-selenocarrageenase extracted from  Vibrio [Cytophaga [Alteromonas [Pseudomonas [As a trace element, selenium is a component of glutathione peroxidase, which protects cell membranes from oxidative damage . CompareVibrio , Cytophaytophaga , Alteromeromonas , Pseudomudomonas . Howeverudomonas .The improvement of living standards and environmental pollution in the 21st century have made people pay more attention to food safety and hygiene. Enzyme preparation used in the process of catalytic processing is a kind of bioactive substance which can be processed into different purities and dosage forms, including immobilized enzymes and cells obtained by immobilization technology . With adPseudoalteromonas sp. Xi13 strain producing κ-selenocarrageenase from the floating ice in Antarctica. In addition, TLC (Thin layer chromatography) analysis showed that the degradation products were mainly disaccharides and tetrasaccharides which displayed antitumor activity against Hela cells by determining the cell viability using Sulforhodamine B (SRB) method [Pseudoalteromonas sp. Xi13 were optimized by using response surface methodology (RSM). In addition, lyophilized and immobilized κ-selenocarrageenases were obtained and their storage stabilities were investigated. Moreover, the selenium content of products degraded by two enzyme preparations was compared to obtain the optimal preparation. The bioenzymatic production of selenium oligosaccharides has the advantages of mild reaction conditions, easy control, and high efficiency. It addresses the problems of high molecular weight and poor water solubility of κ-selenocarrageenan, and lays a foundation for its application as adjuvant for cancer treatment and food additive.In a previous report, we have isolated a ) method . HoweverPolysaccharides can only be stained with Lugol’s iodine solution (a mixture of potassium iodide and iodine components), while oligosaccharides degraded by carrageenases cannot be stained. As shown in Design Expert 8.0.6 software was used to conduct quadratic multiple regression analysis of variance on the experimental results. The quadratic regression equation is the following:R2 value was 0.9730 with a 95% confidence level, indicating that the quadratic polynomial model (Equation (1)) fits the experimental data well. Furthermore, the lack of fit (p = 0.1552 > 0.05) was non-significant, indicating that the model was acceptable and suitable for the optimization of enzyme production in the fermentation of Xi13. The positive linear effects of A (κ-selenocarrageenan concentration), B (Ca2+ concentration), and C (temperature) on Y (enzyme activity) were found to be significant (p < 0.05). All of the quadratics were significant items (p < 0.05), yet those of BC were non-significant (p > 0.05) (The model’s  > 0.05) . The modPedobacter sp. NJ-02 [2+ concentration was found to have significant effects on κ-selenocarrageenase production and cell growth. The optimum concentration of Ca2+ is 3.69 mmol/L, which was consistent with a previous report by Guo et al. [2+ for the production of κ-carrageenase by Thalassospira sp. Fjfst-332 was 0.1 g/L.The Expert Design software was used for further analysis and prediction, and the response surface map and contour map were drawn as p. NJ-02 . When thp. NJ-02 . A high o et al.  showing 2+ , and the temperature 33 °C , respectively. The maximum predictive activity was 8.495 U/mL, increased by about 1.4 times compared with the original enzyme activity (6.079 U/mL). In comparison with 2.14 U/mL of optimized κ-carrageenase production by Cellulosimicrobium cellulans [To sum up, the three factors corresponding to the highest enzymatic activity of the response surface map were 1.6% κ-selenocarrageenan , 3.7 mmol/L Caellulans  investigPseudoalteromonas sp. QY203 was 45 °C [Tamlana sp. HC4 producing carrageenase whose optimum reaction temperature was measured to be 30 °C. Compared with above researches, the thermal stability of κ-selenocarrageenase from Xi13 needs to be further improved.As shown in as 45 °C . Sun et as 45 °C  isolatedPseudoalteromonas sp. ZDY3 [Tamlana sp. HC4 [The pH is an important factor that affects the enzyme activity. As shown in sp. ZDY3  and Taml sp. HC4 .+, Ca2+, K+ and Mg2+. While Ca2+ has the best effect at the same concentration of each ion, which can increase the activity by 72.43%. Mn2+, Zn2+, and Ba2+ at 5.0 mmol/L concentration inhibited the enzyme activity to some degree, while Mn2+ has the strongest inhibitory effect with only 33.21% residual activity. Cu2+ had no obvious effect on enzyme activity. However, Mg2+ and Ba2+ significantly stimulated the κ-carrageenase activity of Pseudoalteromonas porphyrae [2+, Zn2+, Cu2+ and Na+. The reason may be that ions have different effects on the functional domains of proteins due to different structures of kinds of carrageenases.As shown in orphyrae , which iIt can be seen from The relative enzyme activity of κ-selenocarrageenan pre-frozen solution at −20 °C, −80 °C and liquid nitrogen is shown in The optimum catalytic temperature of free enzyme is the same as that of the lyophilized enzyme, and the change trend of their activities is similar a. HoweveThere are various factors such as temperature, pH, air and light that contribute to storage stability of enzyme. After 30 days of storage at −20 °C and 4 °C, the residual enzyme activity was 34.22% and 15.02%, respectively. By contrast, the enzyme activity was completely lost when kept at 25 °C and 37 °C for 30 days a. The spThe morphology of sodium alginate beads with different concentrations of sodium alginate is shown in 2 reached 3% . κ-selenocarrageenan were purchased from Qingdao Pengyang Technology Development Co., Ltd. .Pseudoalteromonas sp. Xi13 was isolated from the floating ice in Antarctica. The strain was cultivated on the 2216E medium  and the κ-selenocarrageenan screening plate .The fermentation broth was centrifuged at 7500 rpm at 4 °C after incubation overnight at 37 °C and then centrifuged at 12,000 rpm at 4 °C for 15 min to obtain the supernatant containing extracellular enzymes. The cell sediment was resuspended with phosphate buffered saline (PBS) solution and broken by ultrasound for 20 min. The cell disruption solution was centrifuged at 12,000 rpm at 4 °C for 15 min to obtain the supernatant containing intracellular enzyme. The κ-selenocarrageenan screening plate was divided into four regions , and then placed at 25 °C for 12 h to investigate κ-selenocarrageenan degradation effect.Using κ-selenocarrageenan as substrate, the activity of κ-selenocarrageenase was measured by DNS  method, and the absorbance was measured at 500 nm . One uniA), Ca2+ concentration (B) and temperature (C), according to the central combination test design principle of RSM [Y. We used 3 factors and 3 levels , β-mercaptoethanol, DTT (dithiothreitol) and PMSF (Phenylmethylsulfonyl fluoride), denaturant , and surfactant (Tween-80) were added to the enzyme solution, and the final concentration was 1.0 mmol/L. The relative enzyme activity, without adding any additive, was referenced as 100% per unit of enzyme activity.With the inactivated enzyme as control, the activity of κ-selenocarrageenase was measured in 0.2% κ-selenocarrageenan solution, at 20, 25, 30, 35, 40, 45, 50, 60, and 70 °C, respectively. To determine the optimal pH of κ-selenocarrageenase, the enzyme solution was mixed with PBS solution with different pH values in equal proportion and placed at 4 °C for 3.0 h to measure the relative enzyme activity. BaThe obtained crude enzyme solution was pre-frozen until the tempreture of it reached −20 °C, −80 °C, respectively. At the same time, the same volume of crude enzyme solution was directly pre-frozen with liquid nitrogen. Then, the above samples were freeze-dried under vacuum for 24 h. The crude enzyme solution was pre-frozen at −80 °C for 6 h, 12 h, 18 h, and 24 h, respectively, and then freeze-dried under vacuum for 24 h to investigate the effect of pre-freezing time on the κ-selenocarrageenase activity. To investigate the effect of thickness of the enzyme solution on κ-selenocarrageenase activity, the crude enzyme solution was pre-frozen at −80 °C for 12 h, and the samples were freeze-dried at a thickness of 3.0, 4.0, 5.0, 6.0, 7.0 mm, respectively. The drying time also affected the κ-selenocarrageenase activity. With the thickness of 4.0, 5.0, and 6.0 mm, respectively, the samples were pre-frozen at −80 °C for 12 h and then freeze-dried for 0, 5, 10, 15, 20, 25, and 30 h, respectively. The activities of enzymic preparations under the above conditions were measured.2 at 10, 20, 30, 40, 50, 70, and 90 min, respectively; then, the enzyme activity was measured.To investigate the effects of temperature, air and light on the stability of the enzyme, the enzyme preparations were stored at −20 °C or 4 °C under different conditions: exposed to or isolated from the air and light, or a combination of the two, respectively. The enzyme activities of free enzyme and lyophilized enzyme were measured at 40 °C for a period of time and at different pH, respectively. The enzyme activity of 0.5 g vacuum-packed lyophilized enzyme was measured every 10 days at −20 °C, 4 °C, 25 °C and 37 °C. Generally, food additives need to meet the requirements of asepsis while ultraviolet irradiation (UV) is usually used for sterilization; thus, the preparations were irradiated under UV with 100 μm/cm2 solution to form smooth pellets. After standing and hardening for 1.0 h, the beads were washed three times with PBS solution. The immobilized enzyme was obtained by removing surface moisture of the beads, then stored at 4 °C.We mixed 3.0% sodium alginate solution and enzyme solution in equal proportion; then, 2.0% glutaraldehyde solution was added to the mixed solution, stirred thoroughly, and incubated for 1.0 h for cross-linking. The above solution was injected into 2.0% CaClThe free enzyme and immobilized enzyme were kept in buffer with different pH at 40 °C for 6.0 h and for 3.0 h, respectively. The free and immobilized enzyme were stored at pH 8.0 at −20 °C and 4 °C for 30 days, respectively. The activities of free enzyme and immobilized enzyme were measured at different time intervals.3, HClO4 andHSO4  at 100 °C for 24 h in a digestion tube until the solution became clear. The digested sample was cooled and diluted to 20 mL with deionized water. The final product was analyzed at 335 nm using UV spectrophotometer  to determine the selenium content. Selenium standard solution (10 µg/mL) was added to 2 mL of 1% o-phenylenediamine hydrochloride solution and diluted to 40 mL with water, respectively. The solution was adjusted to pH 2.0, reacted in the dark for 1.0 h, and extracted with toluene. Standard selenium solutions with different concentrations  were used to develop a standard curve. Inorganic selenium and organic selenium were separated by ethanol precipitation. After the selenium oligosaccharide was dissolved, the content of inorganic selenium in the supernatant was determined. The organic selenium was calculated by subtracting inorganic selenium content from total selenium content.The reaction solution after degradation by the two enzymes was dialyzed against water dialysis bag (cut-off Mw: 3500 Da), concentrated by rotary evaporation, and dried. Dried selenium oligosaccharides in the amount of 1.0 g from all selenite treatment groups were digested with 15 mL of a mixture of HNOPseudoalteromonas sp. Xi13 was optimized using RSM. The maximum κ-selenocarrageenase activity was increased by 1.4 times, reaching 8.416 U/mL under the optimum conditions: 1.6% κ-selenocarrageenan, 3.7 mmol/L Ca2+, and 33 °C. With trehalose as a lyophilized protective agent, the enzyme was freeze-dried under the optimized conditions which are as follows: −80 °C pre-freeze temperature, 12 h pre-freeze time, 2% trehalose, 5 mm thickness of enzyme solution layer. With the increase in drying time, the activity recovery rate of the lyophilized enzyme decreased gradually and tended to be stable at 25 h, which was finally >70%. After 30 days of storage, activity recovery rate of lyophilized enzyme was 36.22%. The selenium content of κ-selenocarrageenan oligosaccharides degraded by the lyophilized enzyme was 43.45 µg/g. With glutaraldehyde as cross-linking agent, the enzyme was immobilized using sodium alginate as carrier under immobilization conditions which are as follows: 3.0% sodium alginate, 0.5% glutaraldehyde, 3.0% CaCl2, 90 min cross-linking time and 120 min fixation time. The activity recovery rate of the immobilized enzyme was 62.83%. The thermal stability and pH tolerance of the immobilized enzyme were significantly better than those of the free enzyme. The enzyme activity remained at 58.28% after reuse four times. The selenium content of κ-selenocarrageenan oligosaccharides degraded by the immobilized enzyme was 47.06 µg/g, 8.3% higher than that degraded by the lyophilized enzyme. All of this work will be useful as a foundation for the industrial production of selenium oligosaccharides.In the present study, the fermentation conditions for producing κ-selenocarrageenase from


The objective of this study was to determine milk fatty acids (FA) from crossbred F1 dairy cows fed on tropical grasses and supplemented with different levels of concentrate. Milk yield and milk composition were not affected while very mild effects were found in milk fatty acids. Overall, this study shows that adding up to 450 g/kg of concentrate to crossbred F1 dairy cows fed on tropical grasses does not have negative effects on milk yield and milk quality. Therefore, under these production conditions, farmers can avoid the use of concentrate, rely on tropical grasses, and reduce feeding costs.p = 0.004), C22:0 (p = 0.031), and C24:0 (p = 0.013). C17:1 cis9 was higher (p = 0.039) with 150 g/kg and lowest with 450 g/kg. C18:1 cis9 was higher (p = 0.042) with 150 g/kg. C18:2n6trans was higher (p = 0.05) with 300 g/kg and lower (p = 0.018) with 450 g/kg. This study shows that adding up to 450 g/kg of concentrate to crossbred F1 dairy cows fed on tropical grasses does not have negative effects on milk yield and milk quality. Therefore, under these production conditions, farmers can rely on tropical grasses and reduce feeding costs.The objective of this study was to determine milk fatty acids from crossbred F1 dairy cows fed on tropical grasses and supplemented with different levels of concentrate. Twelve dairy cows (50% Holstein × 50% Brahman) with 60 days of lactation grazing tropical grasses were assigned to a Switchback design, with three periods of 15 days with different concentrate levels: 0, 150, 300 and 450 g /kg. Milk samples were obtained on the last five days of each experimental period. Milk yield and milk composition were not affected. Cows fed with 300 g/kg of concentrate had higher contents of C15:0 ( Bos taurus and tropical local cattle breeds from Bos indicus was carried out to obtain an animal that is able to cope with temperature and relative humidity [In the tropical regions of Mexico, a first cross-generation F1) from temperate dairy breeds from  from temhumidity . Today, humidity . Howeverhumidity . The need for improved milk yields in F1 dairy cows has led farmers to increase the amount of dietary concentrate but its increase can lead to milk fat depression , digestiThe study was carried out at the Center for Teaching, Research and Extension in Tropical Livestock (C.E.I.E.G.T.) of the National Autonomous University of Mexico (U.N.A.M.) (20°04’ N and 97°03’ W), at an altitude that varies between 99 and 123 m above sea level, with an average annual temperature of 24.5 °C and average annual rainfall of 1991 ± 352 mm.Animal care and procedures were carried out according to the guidelines of the animal care committee of the Universidad Nacional Autónoma de México (project code IT202120). Twelve F1 dairy cows (50% Holstein × 50% Brahman) with 60 days of lactation and average production of 6 kg of milk per day were assigned to a switchback design, with three periods of 15 days with different concentrate levels at 0, 150, 300 and 450 g of DM concentrate/kg of daily milk production over 6 kg/d. ®, Mexico) was supplied according to the treatment during milking time, from 7:30 to 10:00 h, and was made off of 43.3% corn grain; 36.8% sorghum grain, 12.9% soybean meal, 6.0% molasses, and 1% mineral and vitamins premix. The concentrate had 89% dry matter, 18.3% crude protein, 37.4% neutral detergent fiber, 26.2% acid detergent fiber and 9%acid detergent lignin while offered meadows had 20% dry matter, 10.9% crude protein, 72.4% neutral detergent fiber, 38.5% acid detergent fiber and 5.6% acid detergent lignin. The amount of commercial concentrate , with a total grazing area of 46.87 ha. The grass-based pastures were composed of 28% local, mixed native pastures The experimental cows were the last to go into milking to give them time to consume all concentrate. Milking occurred once a day between 07:30 and 10:00 h and milk production was registered as kg/cow/day. The milking parlor was a combination of parallel and herringbone types (“parabone”), capable of milking eight cows at a time.In the last five days of each experimental period, milk production was recorded and pooled samples of 200 mL from each cow were taken for fatty acid analysis. Milk fat separation was carried out using a non-solvent method and the transesterification of FA as reported previously . C13:0 wMilk fatty acids were analyzed using a switchback design for four treatments with three blocks and three periods  and usinijkl is the measured response variable; i—cow; j—treatment; k the period; and l the sequence. Then, μ is an overall mean, δl is the fixed effect due to the sequence of l, β i(l) is a random effect due to the item nesting within the sequence l, αj is a fixed effect due to treatment j, γk is a fixed effect due to period k, αγjk is a fixed interaction effect due to treatment j and period k, and εijkl is the random error.The model included treatment, period and sequence as fixed effects and cows as random effects where Yp < 0.05, using Tukey’s test for means comparison.Milk fatty acid means were subjected to orthogonal polynomial trend analysis . EffectsOverall production performance and milk composition are reported in a companion paper . Brieflyp = 0.004), C22:0 (p = 0.031), and C24:0 (p = 0.013) compared with the rest of the treatments  with 150 g/kg of concentrate and lowest with 450 g/kg (Contents of C17:1 cis9 were highest (450 g/kg . In rumip = 0.042) with 150 g/kg but lower with 0 and 450 g/kg. In humans, intake of oleic acid was reported to be a strategy for managing metabolic disorders such as obesity, insulin resistance, dyslipidemia, and high blood cholesterol [Oleic acid (C18:1 cis9) was higher (lesterol .p = 0.05) with 300 g/kg of concentrate and lower (p = 0.018) with 450 g/kg, while C20:2 was higher with 450 g/kg (Contents of C18:2n6trans were higher (450 g/kg . PreviouPreviously, Bargo et al.  reportedWhen interpreting results from milk fatty acid profiles, it is important to point to the fact that in this study, C13:0 was used as an internal standard and this fatty acid is of rumen microbial origin and can be found in trace amounts in milk. From an analytical perspective, future studies should consider using a different internal standard for this type of sample.In general, this study shows that adding up to 450 g/kg of concentrate to crossbred F1 dairy cows fed on tropical grasses does not have negative effects on milk yield and milk quality, as shown by the lack of differences in milk yield and minor changes in milk fatty acids. Overall, since milk yield and quality were not improved, concentrate supplementation is not needed at least under the conditions of the present study.


Diffuse large B‐cell lymphoma (DLBCL) arising in the spleen, also known as primary splenic DLBCL (PS‐DLBCL), is a rare form of malignant lymphoma. It is defined as a lymphoma confined to the spleen or involving splenic hilar lymph nodes. Here we report a case of PS‐DLBCL with CD30. The patient was a 62‐year‐old who presented with 2 weeks of left flank pain, chills, and abdominal distension. Computed tomography identified an 8‐cm splenic mass with central necrosis interpreted as an abscess. A drain was placed, yielding purulent necrotic material; cytologically, only neutrophils were identified. However, purulent drainage continued for 28 days without resolution, prompting splenectomy. Pathological dissection revealed a multinodular mass with central necrosis. Microscopic examination revealed extensive karyorrhexis, abundant ghosts of large cells, and scattered large cells with pleomorphic, multilobated, and vesicular nuclei with moderately abundant cytoplasm. Immunohistochemical staining revealed large, atypical cells positive for CD20, CD30, CD45, PAX5, MYC (>40%), MUM1 (>30%), and p53 . The large cells were negative for CD3 , CD4, CD5, CD8, CD10, CD15, CD34, BCL2, BCL6, AE1/AE3, S100, HHV8, and ALK. The Ki‐67 proliferation rate was approximately 80% in large cells. Notably, this PS‐DLBCL was positive for CD30, an unusual finding among non‐Hodgkin B‐cell lymphomas, which, coupled with the Reed‐Sternberg‐like morphology, raised the possibility of classic Hodgkin lymphoma. Therefore, we reviewed the literature to confirm the unique features of this large B‐cell lymphoma, its abscess‐like appearance, and its expression of CD30. AlthougPatients with PS‐DLBCL are typically older  and present with left upper quadrant pain, high lactate dehydrogenase levels, and thrombocytopenia , 5. SpleAlthough originally recognized as a marker of classic Hodgkin lymphoma, CD30 is also expressed in a subset of non‐Hodgkin lymphomas, including PTCL, extranodal natural killer/T‐cell lymphomas, cutaneous T‐cell lymphomas, and DLBCL . CD30 poHere, we present a unique case of PS‐DLBCL which presents as a splenic abscess. Interestingly, this lymphoma expressed CD30. We performed a systematic review of the literature and identified only anecdotal similar cases of PS‐DLBCL and discuss their findings.1.19/L, predominantly mature granulocytes at 21.6 × 109/L), normal platelets at 289 × 109/L, and mild normocytic anemia . Computed tomography identified splenomegaly with a hypoenhancing area, concerning for an abscess, as well as a wedge‐shaped infarct . However, the hemoglobin decreased to 9.3 g/dl over the following five days. Patient referred to persistent abdominal pain and developed diarrhea and emesis. After initial surgical evaluation, the patient had a splenic drain placed for purulent material with negative cultures. A fine needle aspiration at that time similarly showed neutrophilic inflammation consistent with an abscess but no malignant cells. During this time the patient was tested positive for severe acute respiratory syndrome coronavirus 2. The patient was discharged, but the tube continued to drain purulent material for 29 days with no change on imaging, prompting a splenectomy.A 62‐year‐old female presented with left flank pain for two weeks followed by the onset of chills, night sweats, and decreased appetite for 1 week. She was prescribed ciprofloxacin by her primary care provider for suspected upper urinary tract infection; however, negative urine cultures and lack of improvement after 2 days led her to present to the emergency department. On physical exam, she was acutely ill‐appearing with moderate abdominal distension and left‐sided tenderness. Laboratory evaluation demonstrated leukocytosis 25 × 10Grossly, the dissected spleen appeared to have a centralized pale tan‐yellow well‐defined soft abscess 8.4 × 8.2 × 6.0 cm) disrupting the hilum, as well as an additional pale‐tan yellow abscess with surrounding hemorrhage (2.4 × 2.0 × 1.3 cm) measuring 0.5 cm from the larger abscess , but negative for MYC rearrangement (8q24 locus) or double fusion of t IgH (14q32) with BCL2 (18q21).Immunohistochemical stains on fixed, paraffin‐embedded tissue sections demonstrated that the large atypical lymphoid cells were positive for CD20 Figure , CD30 F, CD45, PAdditionally, a chromosome microarray was performed at the University of Texas Medical Branch and was consistent with complex DNA copy number aberrations. There are mosaic losses (in 1p) and gains (in 1p and 1q) of chromosome 1, with copy number changes consistent with chromothripsis Figure . There a22.1Hematoxylin and eosin and immunohistochemical (IHC) stains were performed on formalin‐fixed, paraffin‐embedded tissue sections at the University of Texas Medical Branch.The following IHC stains were used: CD4, CD5, CD8, CD10, CD34, PAX‐5, and MUM‐1 ; CD3P, CD15, CD20, CD30, AE1/AE3, BCL‐2, Ki‐67 ; CD45, BCL‐6 and c‐MYC ; P53 and S100 (Ventana/RTU); HHV8 (Cell Marque/RTU); and ALK‐1/CD246 (Dako/RTU). ISH was performed using a probe for EBV‐encoded viral RNAs .2.2BCL6 (3q27), MYC (8q24), and t IgH/BCL2 was performed on fresh tissue.Fluorescence ISH analysis was performed by Vitro Molecular  with probes (Abbott Molecular/Vysis and Cytocell) specific for 2.3Chromosomal microarray analysis was performed using Affymetrix CytoScan HD microarray. This microarray and associated software  are manufactured by Affymetrix  and used by the University of Texas Medical Branch Molecular Diagnostics Laboratory (MDL) for the purpose of identifying DNA copy number gains and losses associated with large chromosomal imbalances.3Given that primary splenic lymphoma constitutes ≤1% of all malignant lymphomas, consistent and detailed characterization is lacking . Few casIGH rearrangements, along with cytogenetic abnormalities including add(7p22), del(8p22), add(19p13), and t [BCL6 rearrangement, and our cytogenetic testing revealed changes not previously reported, including chromothripsis of chromosome 1, segmental losses in 6q, 11p, 16q, and 17p, and gains in chromosomes 6p, 7, 8, and X. These changes may be useful to monitor clonal evolution and progression of splenic lymphoma.Genetic testing for PS‐DLBCL typically reveals monoclonal d t . HoweverStaphylococcus aureus and Klebsiella pneumoniae, but the patient failed to significantly improve with antibiotics. The negative splenic cultures following drainage may have been due to antibiotic treatment , or the splenic abscess may have been ancillary with a simultaneous infection at another site  at presentation.Of particular interest is the presentation of this splenic lymphoma as a continuously‐draining abscess. In the literature review, we identified four reports of splenic lymphoma presenting as an abscess: marginal zone lymphoma , DLBCL , 14, andRegardless, the marked leukocytosis and rapid improvement following IV antibiotics suggest infection in our patient at presentation. Additionally, we speculate that purulent splenic abscess could play an etiological role in PS‐DLBCL development, and more cases need to be studied and described to make it a more prominent conclusion.4In conclusion, we report a striking and unusual case of PS‐DLBCL presenting as a splenic abscess that continuously drained sterile purulent material for 28 days. The literature review demonstrated very rare cases of splenic lymphomas presenting as an abscess, therefore we wanted to highlight the importance of keeping DLBCL as a differential in such a presentation.Paul A. Wadsworth and Kirill A. Lyapichev wrote the manuscript with support from Roberto N. Miranda, Peeyush Bhargava, Dylan Weaver, Michael Norman, Jianli Dong, Vasily Ovechko, Palawinnage V. Muthukumarana, Mathew G. Bayes, and Jayati Mallick. All authors discussed the results and contributed to the final manuscript.The authors declare that they have no conflict of interest.The authors received no financial support for the research, authorship, and/or publication of this article.