KS Group Research Articles

P1231 7-ketositosterol in high-temperature heating oils aggravates colitis by gut microbiota dysbiosis induced-PDLIM3 activation

Abstract Background Diet and gut microbiota alteration are considered as the most important environmental factors triggering inflammatory bowel disease. Phytosterols are rich in edible oils, and can be oxidized to oxyphytosterols during high-temperature cooking. Previous studies have indicated that oxyphytosterols exposure exerted potential adverse effects in cell and animal models. It remains unknown whether oxyphytosterols could promote colonic inflammation. 7-ketositosterol has the highest proportion in oxyphytosterols. Methods The fried and baked foods intake was collected and compared in IBD patients and healthy individuals. 7-ketositosterol was synthesized from β-sitosterol and impacts of 7-ketositosterol on intestinal inflammation was evaluated in colitis mice models. 16S rRNA and transcriptome sequencing were carried out. Furthermore, fecal microbiota transplantation was carried out to evaluate the effect of gut microbiota. Protein interaction of Staphylococcus lentus and PDLIM3 was explored by co-immunoprecipitation. Results The fried and baked foods intake in IBD patients was much more than that of healthy individuals, with higher exposure to oxyphytosterols. 7-ketositosterol (KS) aggravated mice colitis. KS increased the abundance of Actinobacteria (p=0.023). Meanwhile, the abundance of potential pathogens such as Staphylococcus (p=0.004) and Corynebacterium_1 (p=0.003) were increased in KS group, while the abundance of short chain fatty acids producing bacteria were decreased. Transcriptome data showed KS increased the expression of PDLIM3 (p=0.004). PDLIM3 expression was demonstrated to increase in intestinal epithelial cells, and the expression tendency of p-p38 and p-p65 was consistent with PDLIM3. In addition, KS did not exacerbate DSS induced colitis after antibiotic cocktails treatment, and PDLIM3 expression decreased in parallel. The gut microbiota from KS also aggravated DSS induced colitis in antibiotic treated mice. Proteins from Staphylococcus lentus could interact with PDZ domain and promote PDLIM3 expression. Conclusion 7-ketositosterol in high temperature heating oils such as fried foods could aggravate colitis by modulation of gut microbiota. Gut microbiota interacted with PDZ domain, promoted PDLIM3 expression and then activated p38MAPK/NF-κB signaling pathway.

P575 Kono-S anastomosis reduces post-op intervention after ileocolic resection for Crohn’s disease

Abstract Background Ileocolic resection and anastomosis is the most common intestinal operation for Crohn's disease (CD). However, the choice of anastomosis remains a topic of debate. The SUPRME CD randomized trial demonstrated decreased rates of surgical recurrence after Kono-S anastomosis (KSA). We aimed to compare the long-term recurrence-free survival between KSA and stapled side-to-side (SSTS) anastomosis in a retrospective cohort. We hypothesized that KSA was associated with a lower risk of postoperative recurrence requiring intervention than SSTS. Methods We included consecutive patients from a prospectively maintained database who underwent ileocolic anastomosis for terminal ileal CD between 2020 and 2022. Patients with other types of anastomoses were excluded. The short-term postoperative outcomes and interventions for perianastomotic recurrence were compared between the two groups. Results Overall, 174 patients underwent ileocolic anastomosis: KSA, 83 (47.7%) and SSTS, 91 (52.3%). In the KSA group, compared to SSTS, median BMI was lower (24.3 vs. 26.4 kg/m2, p=0.04), median operative time was longer (186 vs. 146 minutes, p<0.001), and patients were more likely to have mesenteric excision (53% vs. 15.4%, p<0.001). There were no significant differences in the short-term postoperative outcomes. After 12 months, 12 (30%) patients in the KSA group and 14 (35%) in the SSTS group developed endoscopic recurrence with a Rutgeerts score ≥i2 (p = 0.81). Compared with the SSTS group, the KS group received more postoperative biologics (65.1% vs. 39.6%, p=0.001) and more frequent medical therapy for perianastomotic recurrence [18/20, 90%] vs. [15/22, 68%]; p=0.14). On multivariable analysis, the KSA (OR 0.09, 95% CI 0.01-0.95, p=0.05) was associated with a lower risk of perianastomotic recurrence requiring surgical and endoscopic intervention. Conclusion We did not observe any statistically significant differences in overall recurrence-free survival between KSA and SSTS. However, the Kono-S anastomosis was independently associated with a decreased rate of endoscopic or surgical intervention for perianastomotic recurrence.

65 Learning Ability in Relation to Everyday Activities in Patients with Korsakoff’s Syndrome, Other Alcohol-Related Cognitive Impairments, or Uncomplicated Alcohol Use Disorder

Objective:Assessment of learning potential in patients with cognitive disorders in individuals with alcohol-related cognitive disorders (including Korsakoff’s syndrome; KS) is highly relevant, as this may help to tailor interventions, guide treatment planning and help to optimize care. However, studies on assessing learning potential or learning ability using neuropsychological assessment in relation to changes in everyday activities during the course of treatment are scarce. In this study we examined whether verbal and visuospatial learning curves could be used as an index of learning ability in relation to everyday activities before and after a treatment program.Participants and Methods:We examined the episodic learning ability of patients with KS (N=137), other alcohol-related cognitive impairments (ARCI; N=164), and uncomplicated alcohol use disorder (AUD; N=49). For this, we calculated the learning curves for the California Verbal Learning Test (CVLT) and the Location Learning Test - Revised (LLT-R) and examined their association with ratings of everyday activities by the patient and his/her professional caregiver using the Patient Competency Rating Scale (PCRS) before and after a 10-12 week treatment program following admission to the Korsakoff Centre.Results:For both verbal and visuospatial memory, the AUD group had a steeper learning curve than the ARCI patients, who in turn had a steeper learning curve than the KS group (p<.01). While the VLGT total score was related to the PCRS in all patient groups (Pearson r>.38, p<.01), this was only the case for the KS group for the LLT-R total score (r>-.29, p<.01). However, the learning curve estimates of both tests were neither related to the PCRS absolute scores (for patients and caregivers, before and after treatment) nor to the APCRS scores during the course of the treatment program.Conclusions:Episodic learning ability, as measured with the learning curves of the CVLT and LLT-R, were unrelated to the patients everyday activity level as measures by the patients themselves or their professional caregiver. The results will be discussed in relation to other tools for assessing the learning potential of cognitively impaired patients, such as dynamic testing.

Assessing the efficacy of four methods established by four parameters in ICL size selection and relevant influencing factors: a prospective cohort study.

To compare the efficacy and relevant influencing factors of four ICL size selection methods established by four different parameters. This prospective study included 60 patients (120 eyes) who underwent bilateral ICL implantation. Patients were equally divided into four groups, and each group used the Parkhurst nomogram based on sulcus-to-sulcus (STS), the manufacturer's Online Calculation & Ordering System (OCOS) nomogram based on white-to-white (WTW), the KS formula based on angle-to-angle (ATA) and the NK formula based on anterior chamber width (ACW) to determine the ICL size. Recorded the vault one month after operation and compared the consistency between STS and WTW, ATA and ACW and their effects on the vault of different groups. The Parkhurst nomogram, OCOS nomogram, KS formula and NK formula determined 86.7%, 70.0%, 83.3% and 66.7% of properly sized ICL, respectively. STS and ATA were correlated (P < 0.05). The mean difference between the STS and WTW, ATA and ACW was -0.37 ± 0.62mm, -0.42 ± 0.53mm and -0.44 ± 0.52mm, respectively. The vault in the OCOS group was negatively correlated with △STS-WTW, and the vault in the NK group was negatively correlated with △STS-WTW, △STS-ATA and △STA-ACW. The vault in the Parkhurst group and KS group was not affected by anterior segment biometry variables. ATA can be served as an alternative parameter to STS, and STS-based Parkhurst nomogram and ATA-based KS formula determined the most appropriate ICL size. When using OCOS nomogram and NK formula to select ICL size, postoperative abnormal vault was associated with a larger difference between STS and other anterior segment parameters.

Calibrated dynamic zonal model DOMA+ using the SCE-UA method – application to atrium temperature distribution prediction

Atriums in modern building designs often represent particular problems related to energy and airflow distribution, such as thermal stratification, local overheating, and uncontrolled contaminant spreading. This study developed a new dynamic zonal model with air-diffuser (DOMA) to predict the airflow and temperature stratification of an atrium, and then it was experimentally calibrated (known as DOMA+) by using the Shuffled Complex Evolution method developed at the University of Arizona (SCE-UA). Two-parameter set groups were considered during the calibration, which are Group One, including the flow coefficient K and the convective heat transfer coefficients for surfaces of the atrium; in Group Two, the flow coefficient K is separated into K-horizontal and K-vertical representing the flow coefficient used in horizontal and vertical directions. The results show that although the Group-One calibrated zonal model with one K is accurate to predict space thermal behavior, an update to two Ks (Group Two) is suggested to improve the zonal model prediction accuracy and quality. The use of the SCE-UA model reveals the inherent limitations and characteristics of the zonal model DOMA and thus indicates how to improve the prediction ability of a zonal modeling approach to advance its development.

First baseline data of the Klinefelter ItaliaN Group (KING) cohort: clinical features of adult with Klinefelter syndrome in Italy

BackgroundKlinefelter syndrome (KS) is frustratingly under-diagnosed. KS have a broad spectrum of clinical features, making it difficult to identify. ObjectiveWe describe KS clinical presentation in a large Italian cohort.DesignThis is the first observational cohort study within a national network, the Klinefelter ItaliaN Group (KING). Primary outcomes were to describe the basic clinical features and the actual phenotype of KS in Italy. Secondary outcomes were to determine age at diagnosis and geographical distribution.MethodsWe performed a basic phenotyping and evaluation of the hormonal values of 609 adult KS patients.ResultsMean age at diagnosis was 37.4 ± 13.4 years. The overall mean testicular size was 3 ml, and 2.5 ml in both testes in untreated KS group. BMI was 26.6 ± 5.8 kg/m2, and 25.5% of KS had metabolic syndrome (MetS). LH and FSH were increased, and mean total testosterone were 350 ± 9.1 ng/dl. A descriptive analysis showed that 329 KS patients were evaluated in Northern Italy, 76 in Central and 204 in Southern Italy. Analysis of variance demonstrated significant statistical differences (p < 0001) between the age at diagnosis of the three geographical groups. Compared with the expected number among male patients matched for age in Italy, only 16% of KS patients received a diagnosis.ConclusionsThese data are the results of the only national database available that collects the clinical and hormonal data of the KS patients, currently referred at the KING centers. In Italy the typical KS patient is overweight, with small testes, and elevated LH and FSH. Only 25.5% of them are diagnosed with MetS. Early detection and timely treatment are mandatory.

Effect of reciprocal crossing Koekoek and Sasso chickens on growth performance, feed efficiency, carcass yield, mortality rate, and genetic components

Reciprocal crosses of Koekoek and Sasso chickens were evaluated for growth performance, feed efficiency, carcass yield, mortality rate, and genetic components using 60 randomly selected 8-wk-cockerels (15 from each mating group) at Jimma University poultry farm. Data were measured for initial and final body weight, feed intake, daily weight gain, and feed conversion ratio. At the end of the study, following a 4-h fasting period, chickens were weighed, humanely slaughtered, and dressed. Carcass weight, dressing percentage and amount and percentage of abdominal fat were determined. Mortality was recorded. Genetic components were calculated. The collected data were analyzed using the General Linear Models (GLM) procedure of the Statistical Analysis System other than mortality rate and genetic components. Mortality rate and genetic components were analyzed using GLIMMIX Procedure of SAS version 9.4. The study revealed that the cross of Koekoek (cockerel) × Sasso (pullet) (KS) performed significantly (P < 0.05) better than the cross of Sasso (cockerel) × Koekoek (pullet) (SK) for final body weight (2615 g vs. 2208 g), feed intake (133 g vs. 93.3 g), daily weight gain (23.7 g vs.18.3 g), empty carcass weight (2006 g vs. 1604 g), and dressing percentage (77.1 g vs. 72.6 g). Further, the KS group survived significantly (P < 0.05) better than SK with the lowest mortality rate during the experimental period (0.004 vs. 0.009). KS has performed positively and highly in heterosis percentage (H %) for all parameters while SK has performed negatively for former traits. Thus, the crossing of Koekoek cockerels with Sasso pullets should be preferred to its reciprocal cross to obtain higher levels of heterosis for growth performance, feed efficiency, and carcass yield.

Cortical gray matter structure in boys with Klinefelter syndrome

Klinefelter syndrome (KS, 47,XXY) is a common sex chromosome aneuploidy in males that is associated with a wide range of cognitive, social and emotional characteristics. The neural bases of these symptoms, however, are unclear. Brain structure in 19 pre- or early-pubertal boys with KS (11.5 ± 1.8 years) and 22 typically developing (control) boys (8.1 ± 2.3 years) was examined using surface-based analyses of cortical gray matter volume, thickness and surface area. Boys in the KS group were treatment-naïve with respect to testosterone replacement therapy. Reduced volume in the insula and dorsomedial prefrontal cortex was observed in the KS relative to the TD group, as well as increased volume in the parietal, occipital and motor regions. Further inspection of surface-based metrics indicated that whereas KS-associated increases in volume were driven by differences in thickness, KS-associated reductions in volume were associated with decreases in surface area. Exploratory analyses additionally indicated several correlations between brain structure and behavior, providing initial support for a neural basis of cognitive and emotional symptoms of this condition. Taken together, these data add support for a neuroanatomical phenotype of KS and extend previous studies through clarifying the precise neuroanatomical structural characteristics of that give rise to volumetric alterations.

Kıl Keçilerinde Üreme Mevsiminde Kızgınlık Senkronizasyonu Uygulamasının Döl Verimi ve Doğum Ağırlığı Üzerine Etkisi

Bu çalışma ile Kıl keçilerinde üreme mevsimi içinde kızgınlık senkronizasyon uygulamasının döl verimi üzerine etkisinin belirlenmesi amaçlanmıştır. Hayvan materyali olarak 3-5 yaş aralığında toplam 200 baş kıl keçisi kullanılmıştır. Keçiler eşit sayıda olacak şekilde iki gruba ayrılmışlardır (Kontrol grubu, KG n=100; Kızgınlık senkronizasyon grubu, KS, n=100). KS grubuna, 30 mg flugeston asetat içeren sünger takılmış ve 12 gün sonra çıkarılarak hayvan başına 500 IU PMSG kas içi uygulanmıştır. 4 gün süreyle kıl tekeleri kullanılarak çiftleştirme işlemi gerçekleştirilmiştir. Teke katım işlemi her iki grupta da aynı anda başlatılıp 4 gün sonra sonlandırılmıştır. Tekiz doğum oranı KG‘da %72, KS grubunda ise %35 olarak gerçekleşmiştir. Teke altı keçi başına doğan oğlak sayısı ve doğuran keçi başına doğan oğlak sayısı sırasıyla KG‘da 1,20 ve 1,59, KS grubunda ise 1,25 ve 1,64 olarak tespit edilmiştir. Sonuç olarak, Kıl keçilerinde üreme mevsimi içinde kızgınlık senkronizasyon uygulaması döl verimini iyileştirmiştir.

Tracking plasma KRAS mutations (mu) in lung adenocarcinoma (LUAC) patients (p) and branching evolution.

9055 Background: KRAS m in LUAC p are recalcitrant to therapy. In mice models and p, STK11 m confer poor prognosis. Patients with KRAS, or KRAS with TP53 m, benefit from immunotherapy (IO). We used a cell free DNA (cfDNA) sequencing platform to sub-group 53 LUAC p with KRAS m from the Spanish Lung Liquid versus Invasive biopsy Program (SLLIP, NCT03248089), according to the coexistence of TP53 and STK11 m. We evaluated the treatment outcome in the KRAS subgroups and we explored the mutational evolution at 2 weeks (w), and at the end of the study (EOS). Methods: SLLIP was a multi-center observational study in patients with treatment-naïve metastatic LUAC. Genotyping, with a clinically validated cfDNA assay (Guardant360) was performed at 3 time points: before start of treatment, at 2 w, and upon progression or at 12 months (mo). Oncoprints were constructed based on mutation status and variant allele frequency (VAF). Results: 53 p with KRAS alterations were included. 36 male; median age 59; 46 current/ex-smokers; 64% received 1st line platinum-based chemotherapy (CT), 4% platinum-based CT+IO, 8% platinum-based CT plus bevacizumab, and 24% other or no therapy. 13 p had only KRAS m (K-only group), 25 p had KRAS + TP53 m (KP group), and 15 p had KRAS + STK11 with or without TP53 m (KS group). Median progression-free survival was 3.5 mo for all 53 p, 4.8 mo for the K-only group, 4.4 mo for the KP group, and only 2.6 mo for the KS group (p = 0.05 for KS versus K-only). The average VAF for K-only, KP, and KS groups at EOS were 6.4%, 9.7%, and 46%, respectively. When looking at p with cfDNA analysis at the three time points, the following were observed: In the K-only group, 25% lost KRAS m at 2 w and 50% at EOS. 50% and 75% gained TP53 m, at 2 w and EOS, respectively. None gained STK11 at the 2 time points. In the KP group, 40% lost KRAS m at 2 w but all had KRAS m at EOS. 20% and 10% lost TP53 m, at 2 w and EOS, respectively. 10% gained STK11 m at the 2 time points. In the KS group, 33% lost KRAS m at the 2 time points. All and 33% lost STK11 m at 2 w and EOS, respectively. 66% gained TP53 at the 2 time points. Conclusions: Subgroups of KRAS m traced in cfDNA confirm dismal prognosis to 1st line therapies. These findings prompt us to tailor IO for K-only or KP subgroups. For p with STK11 m, restoration of the STK11 function is warranted. Clinical trial information: NCT03248089.

Molecularly confirmed Kabuki (Niikawa-Kuroki) syndrome patients demonstrate a specific cognitive profile with extensive visuospatial abnormalities.

Kabuki (Niikawa-Kuroki) syndrome (KS) is caused by disease-causing variants in either of two components (KMT2D and KDM6A) of the histone methylation machinery. Nearly all individuals with KS have cognitive difficulties, and most have intellectual disability. Recent studies on a mouse model of KS suggest disruption of normal adult neurogenesis in the granule cell layer of the dentate gyrus of the hippocampus. These mutant mice also demonstrate hippocampal memory defects compared with littermates, but this phenotype is rescued postnatally with agents that target the epigenetic machinery. If these findings are relevant to humans with KS, we would expect significant and disproportionate disruption of visuospatial functioning in these individuals. To test this hypothesis, we have compiled a battery to robustly explore visuospatial function. We prospectively recruited 22 patients with molecularly confirmed KS and 22 IQ-matched patients with intellectual disability. We observed significant deficiencies in visual motor, visual perception and visual motor memory in the KS group compared with the IQ-matched group on several measures. In contrast, language function appeared to be marginally better in the KS group compared with the IQ-matched group in a sentence comprehension task. Together, our data suggest specific disruption of visuospatial function, likely linked to the dentate gyrus, in individuals with KS and provide the groundwork for a novel and specific outcome measure for a clinical trial in a KS population.

UJI AKTIVITAS JUS BUAH JAMBU BIJI BERDAGING MERAH (Psidium guajava L.) TERHADAP PENINGKATAN TROMBOSIT PADA MENCIT (Mus musculus L.) YANG DIINDUKSI KLORAMFENIKOL

Thrombocytopenia is the most common hemostatic disorder acquired in dogs and can be a life-threatening. Guava fruit and leaves extract are believed to improve thrombocyte counts. The purpose of this study was to prove that administration of guava juice could improve thrombocyte count in mice that have thrombocytopenia after induced by chloramphenicol. We were used 24 mice as samples divided into 4 groups : KS group (given aquades for 12 days), KN group (induced by chloramphenicol for 7 days, then aquades on next 5 days), group P1 (induced by chloramphenicol for 7 days then guava juice 5 g/ml concentration on 5 next day); and group P2 (induced by chloramphenicol for 7 days then guava juice 10 g/ml concentration on the next 5 days). Thrombocyte count was measured three times, before treatment (day 0), after chloramphenicol induction (day 7), and after guava juice (day 12). Data of thrombocyte count was analyzed using one way Anova and Post Hoc LSD test. The results showed that the guava juice (Psidium guajava L.) concentration of 5 g/ml and 10 g/ml had significant effect (p &lt;0,05) to increase platelet numbers in mice after induced by chlorampenicol dose 30mg/30grBW.

UJI AKTIVITAS JUS BUAH JAMBU BIJI BERDAGING MERAH (Psidium guajava L.) TERHADAP PENINGKATAN TROMBOSIT PADA MENCIT (Mus musculus L.) YANG DIINDUKSI KLORAMFENIKOL

Thrombocytopenia is the most common hemostatic disorder acquired in dogs and can be a life-threatening. Guava fruit and leaves extract are believed to improve thrombocyte counts. The purpose of this study was to prove that administration of guava juice could improve thrombocyte count in mice that have thrombocytopenia after induced by chloramphenicol. We were used 24 mice as samples divided into 4 groups : KS group (given aquades for 12 days), KN group (induced by chloramphenicol for 7 days, then aquades on next 5 days), group P1 (induced by chloramphenicol for 7 days then guava juice 5 g/ml concentration on 5 next day); and group P2 (induced by chloramphenicol for 7 days then guava juice 10 g/ml concentration on the next 5 days). Thrombocyte count was measured three times, before treatment (day 0), after chloramphenicol induction (day 7), and after guava juice (day 12). Data of thrombocyte count was analyzed using one way Anova and Post Hoc LSD test. The results showed that the guava juice (Psidium guajava L.) concentration of 5 g/ml and 10 g/ml had significant effect (p &lt;0,05) to increase platelet numbers in mice after induced by chlorampenicol dose 30mg/30grBW.

Local analgesic effect of a bioadhesive barrier-forming oral liquid in cancer patients with oral mucositis caused by chemotherapy and/or radiotherapy: a randomized multicenter, single-use, positive-controlled, open-label study.

ObjectiveCAM2028 (Episil®; Camurus AB, Lund, Sweden) is a liquid for use in the oral cavity to treat various pains associated with mouth injuries. Upon contact with the swollen oral mucosa, the oral liquid forms a thin protective film that acts as a mechanical barrier to relieve pain. This study was the first in China to evaluate the local analgesic effect of oral liquid in cancer patients who developed oral mucositis following chemotherapy and/or radiotherapy.MethodsA total of 60 patients were randomized in a 1:1 ratio to the CAM2028 group (the pump device was firmly pressed three times and the fluid was distributed to the painful area of the oral cavity) or KS (a mucoadhesive oral wound rinse, Kangsu™; Luye Pharmaceutical Co. Ltd, Nanjing, China) group (5 mL of the oral rinse was poured into and kept in the oral cavity for at least 1 minute). The primary endpoint was the area under the oral mucosal pain score–time curve (AUC) within 6 hours of treatment in the trial and control groups. Medical device adverse events were assessed according to the National Cancer Institute’s Common Terminology Criteria for Adverse Events, version 4.0. Statistical analyses were performed using the chi-squared test (Fisher’s exact test), independent-samples t-test, and analysis of covariance.ResultsSixty patients were included in the per-protocol set population analysis. The average (mean ± SD) 6-hour AUC of the CAM2028 group and the KS group was 14.20±10.29 and 24.46±14.15, respectively. The difference between the groups was statistically significant (P=0.0022). The incidence of adverse events in the trial group and the control group was 16.67% and 30.0%, respectively, and there was no statistical difference.ConclusionsCAM2028 displayed an efficacious local analgesic effect in cancer patients who developed oral mucositis following chemotherapy and/or radiotherapy. The results demonstrated its potential value in clinical applications.

A diagnostic germ cell score for immature testicular tissue at risk of germ cell loss.

Can a systematic scoring procedure provide crucial information on the status of highly heterogeneous immature human testicular tissues in the context of cryopreservation for fertility preservation? We developed a systematic histological score as a novel diagnostic tool which differentiates the patient cohort according to the status of germ cell differentiation and number of spermatogonia (normal, diminished and absent), and which could be relevant in the fertility clinic. Cryopreservation of testicular tissue of immature boys is currently considered the option for future fertility restoration. However, experimental techniques for the derivation of sperm as well as valid diagnostic scoring of these immature testis tissues are not yet reported. Testicular tissues of 39 patients (aged 2-20 years) who attended our clinic for cryopreservation between 2010 and 2015 were analyzed to determine the variability of testicular tissue composition, germ cell numbers and differentiation status. Human testicular tissue samples were divided into three groups. Group NT included patients suffering from diseases which do not directly affect the testes (n = 6; aged 6-14 years), group AT included patients suffering from diseases that directly affect the testes (n = 14; 2-17 years), and group KS (Klinefelter patients, n = 19; 12-20 years). Based on immunohistochemical stainings for MAGEA4, the differentiation status as well as the numbers of gonocytes, spermatogonia and spermatocytes were determined. Testicular tissue samples from the NT group contained a mean of 100.3 spermatogonia/mm3 (×103). Highly heterogeneous and significantly lower mean numbers of spermatogonia were scored in testes from boys after cytotoxic exposures or with pre-existing disease (AT group: 35.7 spermatogonia/mm3 (×103); KS group: 1.8 spermatogonia/mm3 (×103)). In addition, the germ cell differentiation status was determined and revealed tissues with either spermatogonia and gonocytes, only spermatogonia, spermatogonia and spermatocytes, or all three germ cell types were present. Based on spermatogonial numbers and differentiation status, we developed a germ cell score which we applied to each individual patient sample. Normal human testicular tissue samples are difficult to obtain for ethical reasons and the sample numbers were small. However, six such samples provide a valid baseline for the normal situation. Fertility preservation of immature male tissues is an emerging field and is currently offered in many specialized centers worldwide. Our diagnostic germ cell score delivers an easily applicable tool, facilitating patient counseling and thus ensuring comparability between the centers with regard to future studies. This study was supported by the Funding Initiative: Translational Research, Ministry of Innovation, Science and Research, Federal State of North Rhine Westphalia (z1403ts006). The authors declare that they do not have competing financial interests.

Premedication with fentanyl-midazolam improves sevoflurane anesthesia for surgical intervention in laboratory mice.

Balanced anesthesia allows for a reduced dosage of each component, while inducing general anesthesia of sufficient depth with potentially fewer side effects. Here, we compare two anesthetic protocols combining sevoflurane anesthesia with pre-medication (ketamine [K] or fentanyl-midazolam [FM]) to a sevoflurane monoanesthesia (S) concerning their ability to provide reliable anesthesia suitable for moderate surgery in laboratory mice. Twenty-one female C57BL/6J mice assigned randomly to one of three protocols underwent a 50-min anesthesia and a sham embryo transfer. Heart rate and core body temperature were continuously recorded by telemetry intra-operatively and for three days pre- and three days post-surgery. Intra-operative respiratory rate was determined by counting thorax movements. Body weight, food, and water intake were measured daily for three days pre- and three days post-surgery. The heart rate in the KS group remained at baseline level throughout the 50-min of anesthesia and surgery. FMS caused a lower heart rate and S alone caused a higher heart rate compared to baseline values. Intra-operative body temperature was at baseline levels in all groups. A decreased respiratory rate was observed in all groups compared to baseline values obtained from resting mice of the same strain, sex and age-distribution. Surgical stimuli induced no significant changes in heart rate and respiratory rate in the KS or FMS group but significant respiratory alteration in the S group compared to baseline values obtained 10 s before applying the stimulus. Post-operative heart rate was above baseline values in all groups; with a significant deviation in the S group. There were no changes in body weight, food, and water intake. In summary, FMS was superior to KS and S for moderate surgery in laboratory mice resulting in less inter-individual variability in response to painful stimuli. Fentanyl and midazolam reduced the depressant effect of sevoflurane on the respiratory rate and the negative post-anesthetic effects on the heart rate. Impact statement With approximately 65 million animals used per year mice are still the most prevalent laboratory mammal species worldwide. In course of biomedical research projects approximately 40% of mice will undergo one or more short or long-term anesthesia. Sufficient anesthetic depth, cardiovascular stability, adequate analgesia, and short recovery times are essential requirements of anesthetic protocols to meet animal welfare. Anesthesia in mice and rats are only to be performed by personnel with appropriate basic training and experience. However, more and more adapted and advanced anesthetic protocols, required to answer very specific scientific questions, often exceed the skills acquired through basic training and present a major challenge to researchers. It is therefore of great importance to further develop and evaluate safe and reliable anesthetic protocols as presented in this study to provide new perspectives on this challenging problem.

Genotypic distribution of HHV-8 in AIDS individuals without and with Kaposi sarcoma: Is genotype B associated with better prognosis of AIDS-KS?

AIDS-associated Kaposi's sarcoma (AIDS-KS) caused by human herpes virus 8 (HHV-8) is the most severe and resistant form of KS tumor. Our aim was to verify whether there is an association between HHV-8 variability and development of AIDS-KS in Brazil by comparing the HHV-8 variability between individuals without and with KS. Saliva samples and blood, when available, were analyzed by polymerase chain reaction (PCR) techniques for detection of the fragments of ORF K1 of HHV-8, which were then genotyped and analyzed regarding the genetic variability. Our study described 106 positive cases for HHV-8 in the saliva from 751 AIDS patients without previous KS. In addition, we performed a phylogenetic analysis of HHV-8 in 34 of the 106 AIDS patients without KS and in 33 of the 37 patients with active KS. The distribution of HHV-8 genotypes A, B, C, and F in AIDS individuals was indistinguishable by comparing non-KS and KS groups, as well as regarding ethnicity. Considering the KS group, genotype B was associated with better prognosis of KS tumor. Interestingly, we found a particular profile of diversity within clade C and 2 recombinant patterns of HHV-8 in the saliva of AIDS individuals without KS. We emphasize the need to achieve standard genotyping protocol for ORF K1 amplification, thus allowing for substantial detection of HHV-8 variants. Our findings can shed light on the role of HHV-8 variability in the pathogenesis of AIDS-KS.

Is the protein expression window during testicular development affected in patients at risk for stem cell loss?

Is the protein expression window during testicular development affected in prepubertal patients at risk for stem cell loss? Nuclear ubiquitin carboxyl-terminal esterase L1 (UCHL1) expression in Sertoli cells and interstitial expression of inhibin α (INHA), sex-determining region Y-box 9 (SOX9) and steroidogenic acute regulatory protein (STAR) was affected in patients with Klinefelter syndrome. Some patients undergoing testicular tissue banking have already been treated before the testis biopsy is taken. These treatments include chemotherapy or hydroxyurea, which can have an influence on the stem cell number and function. A germinal loss occurs in Klinefelter patients, but its cause is currently unknown. Parrafin-embedded testicular tissue from 5 fetuses, 25 prepubertal patients and 5 adults was used to characterize the spatial and temporal distribution of different testicular marker proteins during testicular development. Expression of the markers was evaluated in germ cells, Sertoli cell and interstitial cells. The integrity of this time window was analyzed in patients at risk for germ cell loss: patients treated with hydroxyurea (n = 7), patients treated with chemotherapy (n = 6) and patients affected by Klinefelter syndrome (n = 5). Immunohistochemistry was performed in normal fetal, prepubertal and adult testicular tissue to set up a timeline for the expression of melanoma antigen family A4 (MAGE-A4), ubiquitin carboxyl-terminal esterase L1 (UCHL1), octamer-binding transcription factor 4 (OCT4), stage-specific embryonic antigen-4 (SSEA4), homeobox protein NANOG, INHA, anti-Müllerian hormone, androgen receptor (AR), SOX9 and STAR. The established timeline was used to evaluate whether the expression of these markers was altered in patients at risk for germ cell loss (patients treated for sickle cell disease (hydroxyurea) or cancer (chemotherapy) and patients with Klinefelter syndrome). A protein expression timeline was created using different markers expressed in different testicular cell types. Less positive tubules and less positive cells per tubule were observed for MAGE-A4 and UCHL1 expression in the KS compared with the non-treated group (P < 0.01). Higher nuclear UCHL1 Sertoli cell expression was observed in the KS group compared with the non-treated group (P < 0.05). Higher interstitial expression of INHA (P < 0.05), SOX9 (P < 0.01) and STAR (P < 0.05) was observed in KS compared with the non-treated group. Important age variations exist in the prepubertal groups. Therefore, data were represented in three age groups. However, owing to the limited access to prepubertal tissue, no statistical comparison was possible between these groups. For the Klinefelter group, tissue was only available from patients older than 12 years. The expression timeline can add knowledge to the process of spermatogenesis and be used to evaluate altered protein patterns in patients undergoing potentially gonadotoxic treatments, to monitor spermatogenesis established in vitro and to unravel causes of germ cell loss in Klinefelter patients.

Improvement of Large Animal Model for Studying Osteoporosis

Abstract The aim of the study was to determine the impact of steroidal medications on the structure and mechanical properties of supporting tissues of sheep under experimentally-induced osteoporosis. A total of 21 sheep were used, divided into three groups: a negative control (KN) (n = 3), a positive control (KP) (n = 3) with ovariectomy, and a steroidal group (KS) (n = 15) with ovariectomy and glucocorticosteroids. All animals were kept on a low protein and mineral diet and had limited physical activity and access to sunlight. Quantitative computed tomography was the examination method. The declines in the examined parameter values in the KS group were more than three times higher than in the KN group. The study suggests that a glucocorticosteroidal therapy accelerates and intensifies processes taking place in the course of osteoporosis. The combination of glucocorticosteroids with ovariectomy, a restrictive diet, limited physical activity, and no access to sunlight leads to a decrease in radiological bone density.

Discourse-level Prosody Produced by Korean Learners of English

This study investigated (1) whether Korean learners of English use discourse-level prosody in L2 production as native speakers of English do, and (2) whether discourse-level prosody is also found in the Korean language, as is evident in the prosody of native speakers of English. The study compared the production of the same 15 sentences in two types of reading materials, sentence-level and discourse-level. This study analyzed the onset pitch, sentence mean pitch and pause length to examine the paratone (intonational paragraph) realization in discourse-level speech. The results showed that in L2 discourse-level prosody, the Korean speakers were limited in displaying paratone and did not made significant difference between sentence-level and discourse-level prosody. On the other hand, in L1 discourse-level text, both English and Korean participants demonstrated paratone using pitch. However, there were differences in using prosodic cues between two groups. In using pauses, the ES group paused longer before both the orthographically marked and not marked topic sentences. The KS group paused longer only before the orthographically marked topic sentence in both L1 and L2 text reading. In the comparison of sentence-level and discourse-level prosody, the topic sentences were marked by different prosodic cues. English participants used higher sentence mean pitch, and the Korean participants used higher onset pitch.