Genome survey and development of polymorphic microsatellite loci for Sillago sihama based on Illumina sequencing technology.

In this study, we first conducted a genome survey assay for Sillago sihama by Illumina sequencing platform, and then developed 15 polymorphic microsatellite loci in a wild population. A total of 129.46 Gb raw data were obtained, of which 115.07 Gb were clean data, with a sequencing depth of 179.3-folds. This genome was estimated to be 522.6 Mb in size, with the heterozygosity, repeat content and GC content being 0.63%, 21% and 44%. A total of 630,028 microsatellites were identified from the genome, of which, dinucleotide repeat was the most abundant (56.80%), followed by mononucleotide repeat (30.23%). Furthermore, 60 pairs of primers were designed and synthesized based on microsatellite sequences, of which 15 were polymorphic in a wild population. A total of 91 alleles were found, with an average of 6.07 per locus. Number of alleles, observed and expected heterozygosity per locus ranged from two to 13, from 0.250 to 0.862, and from 0.396 to 0.901, respectively. Twelve loci were highly informative (PIC > 0.5), and the others were medium informative (0.25 < PIC < 0.5). Seven loci deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.0033). No significant linkage disequilibrium was detected between loci pairs. This study provided a large number of genomic resources and 15 polymorphic microsatellite loci that should be helpful for the further genetic studies in S. sihama.

Distinct but overlapping binding sites of agonist and antagonist at the relaxin family peptide 3 (RXFP3) receptor.

The relaxin-3 neuropeptide activates the relaxin family peptide 3 (RXFP3) receptor to modulate stress, appetite, and cognition. RXFP3 shows promise as a target for treating neurological disorders, but realization of its clinical potential requires development of smaller RXFP3-specific drugs that can penetrate the blood-brain barrier. Designing such drugs is challenging and requires structural knowledge of agonist- and antagonist-binding modes. Here, we used structure-activity data for relaxin-3 and a peptide RXFP3 antagonist termed R3 B1-22R to guide receptor mutagenesis and develop models of their binding modes. RXFP3 residues were alanine-substituted individually and in combination and tested in cell-based binding and functional assays to refine models of agonist and antagonist binding to active- and inactive-state homology models of RXFP3, respectively. These models suggested that both agonists and antagonists interact with RXFP3 via similar residues in their B-chain central helix. The models further suggested that the B-chain Trp27 inserts into the binding pocket of RXFP3 and interacts with Trp138 and Lys271, the latter through a salt bridge with the C-terminal carboxyl group of Trp27 in relaxin-3. R3 B1-22R, which does not contain Trp27, used a non-native Arg23 residue to form cation-π and salt-bridge interactions with Trp138 and Glu141 in RXFP3, explaining a key contribution of Arg23 to affinity. Overall, relaxin-3 and R3 B1-22R appear to share similar binding residues but may differ in binding modes, leading to active and inactive RXFP3 conformational states, respectively. These mechanistic insights may assist structure-based drug design of smaller relaxin-3 mimetics to manage neurological disorders.

Finding the Right Blend of Technologically Enhanced Learning Environments: Randomized Controlled Study of the Effect of Instructional Sequences on Interprofessional Learning.

BACKGROUND: With the availability and capabilities of varied technologically enhanced learning activities, the blended learning approach has become increasingly popular in interprofessional education. The combined use of different technologically enhanced learning activities has not been fully examined, particularly to determine the effects of instructional sequences for effective learning outcomes. OBJECTIVE: The objective of this study was to investigate whether the instructional sequences of a blended learning approach can improve students' learning outcomes on interprofessional competencies. METHODS: A randomized controlled study was conducted with 40 interprofessional health care teams. These teams undertook three technologically enhanced learning activities-Web-based instruction (WI), virtual reality (VR), and simulation exercise (SE)-after random assignment to three groups based on three different instructional sequences (WI-VR-SE, WI-SE-VR, and SE-WI-VR). Pretests and posttests were conducted to evaluate the students' learning outcomes on interprofessional competencies. RESULTS: A total of 198 participants from the three groups completed the questionnaires. All three groups reported significant improvement in their levels of self-efficacy (P<.05) and attitudes (P<.001) toward interprofessional team care about 1 month after the interprofessional learning activity. Although no significant difference was found (P=.06) between the WI-VR-SE and WI-SE-VR groups in the self-efficacy posttests, participants in the SE-WI-VR group reported significantly lower (P<.05) posttest scores than those in the WI-SE-VR group. The majority of the participants (137/198, 69.1%) selected the instructional sequence "WI-VR-SE" as their top preference. CONCLUSIONS: This study shows that the instructional sequence of a blended learning approach can have a significant impact on students' learning outcomes. The learning of concepts from WI followed by problem-solving activity in the SE was found to be a more effective learning sequence than the reverse sequence. We recommend that future studies focus on scaffolding students' learning when planning instructional sequences for technologically enhanced learning activities within blended learning environments.

The Effects of the Organic Flame-Retardant 1,2-Dibromo-4-(1,2-dibromoethyl) Cyclohexane (TBECH) on Androgen Signaling in Human Prostate Cancer Cell Lines.

The effects of the organic flame retardant 1,2-Dibromo-4-(1,2-dibromoethyl) cyclohexane (TBECH) on androgen receptor target gene expression were examined in the human LNCaP prostate cancer cell line. While γ-/δ-TBECH alone led to a significant increase in prostate-specific antigen (PSA) mRNA accumulation, both the α-/-TBECH and γ-/δ-TBECH mixtures repressed androgen-inducible PSA mRNA and protein accumulation in human LNCaP cells. Thus, we hypothesize that isomeric mixtures of TBECH may act as partial agonists of the androgen receptor.

Synthesis and pharmacological characterization of a europium-labelled single-chain antagonist for binding studies of the relaxin-3 receptor RXFP3.

Relaxin-3 and its endogenous receptor RXFP3 are involved in fundamental neurological signalling pathways, such as learning and memory, stress, feeding and addictive behaviour. Consequently, this signalling system has emerged as an attractive drug target. Development of leads targeting RXFP3 relies on assays for screening and ligand optimization. Here, we present the synthesis and in vitro characterization of a fluorescent europium-labelled antagonist of RXFP3. This ligand represents a cheap and safe but powerful tool for future mechanistic and cell-based receptor-ligand interaction studies of the RXFP3 receptor.

'Let's talk about sex' - A Knowledge, Attitudes and Practice study among Paediatric Nurses about Teen Sexual Health in Hong Kong.

AIMS AND OBJECTIVES: To explore the knowledge, attitudes and practices of paediatric nurses in Hong Kong towards adolescent sexual health issues. BACKGROUND: In Hong Kong, teens are becoming more sexually permissive. As a result, early sexual activity, Sexually transmitted diseases (STD) and unplanned pregnancies among adolescents are increasing. Paediatric nurses are potentially excellent sexual health educators; however, studies in other countries have reported that nurses have inadequate knowledge and skills about sexual health. Little is known about the knowledge, attitudes and practices of nurses in Hong Kong related to teen sexual health. DESIGN: This is a cross-sectional survey study. The survey was developed after an extensive literature review and partially adapted from previously validated questionnaires on nursing needs, knowledge, attitude and practice. METHODS: An anonymous self-administered questionnaire in Chinese was distributed to 500 nurses in Hong Kong attending a local paediatric conference. Participants (n = 394) were recruited using convenience sampling methods. Survey data were analysed using descriptive statistics, correlation tests and logistic regression analyses. RESULTS: The majority of nurses had a high awareness of the importance of sexual healthcare, but rarely felt knowledgeable or comfortable discussing sexual health issues with adolescents. Higher comfort level was associated with higher frequency of practice with an average adjusted odds ratios of 2·64. Inadequate or lack of training (39·4%) was the most significant barrier in providing adolescent sexual health screening and counselling. CONCLUSION: Nurses' clinical practices towards adolescent's sexual health issues were influenced by their perceived comfort level followed by their self-ranked knowledge and training experience. Further specific training on communication, counselling and general sexual health should be provided to nurses in Hong Kong. RELEVANCE TO CLINICAL PRACTICE: Nurses' comfort level was the most important factor influencing their clinical practice with teens. Further specific training should be provided to nurses in Hong Kong to improve adolescent sexual health.

Age at cancer diagnosis by breed, weight, sex, and cancer type in a cohort of more than 3,000 dogs: Determining the optimal age to initiate cancer screening in canine patients.

The goal of cancer screening is to detect disease at an early stage when treatment may be more effective. Cancer screening in dogs has relied upon annual physical examinations and routine laboratory tests, which are largely inadequate for detecting preclinical disease. With the introduction of non-invasive liquid biopsy cancer detection methods, the discussion is shifting from how to screen dogs for cancer to when to screen dogs for cancer. To address this question, we analyzed data from 3,452 cancer-diagnosed dogs to determine the age at which dogs of certain breeds and weights are typically diagnosed with cancer. In our study population, the median age at cancer diagnosis was 8.8 years, with males diagnosed at younger ages than females, and neutered dogs diagnosed at significantly later ages than intact dogs. Overall, weight was inversely correlated with age at cancer diagnosis, and purebred dogs were diagnosed at significantly younger ages than mixed-breed dogs. For breeds represented by ≥10 dogs, a breed-based median age at diagnosis was calculated. A weight-based linear regression model was developed to predict the median age at diagnosis for breeds represented by ≤10 dogs and for mixed-breed dogs. Our findings, combined with findings from previous studies which established a long duration of the preclinical phase of cancer development in dogs, suggest that it might be reasonable to consider annual cancer screening starting 2 years prior to the median age at cancer diagnosis for dogs of similar breed or weight. This logic would support a general recommendation to start cancer screening for all dogs at the age of 7, and as early as age 4 for breeds with a lower median age at cancer diagnosis, in order to increase the likelihood of early detection and treatment.

Cancer detection in clinical practice and using blood-based liquid biopsy: A retrospective audit of over 350 dogs.

BACKGROUND: Guidelines-driven screening protocols for early cancer detection in dogs are lacking, and cancer often is detected at advanced stages. HYPOTHESIS/OBJECTIVES: To examine how cancer typically is detected in dogs and whether the addition of a next-generation sequencing-based "liquid biopsy" test to a wellness visit has the potential to enhance cancer detection. ANIMALS: Client-owned dogs with definitive cancer diagnoses enrolled in a clinical validation study for a novel blood-based multicancer early detection test. METHODS: Retrospective medical record review was performed to establish the history and presenting complaint that ultimately led to a definitive cancer diagnosis. Blood samples were subjected to DNA extraction, library preparation, and next-generation sequencing. Sequencing data were analyzed using an internally developed bioinformatics pipeline to detect genomic alterations associated with the presence of cancer. RESULTS: In an unselected cohort of 359 cancer-diagnosed dogs, 4% of cases were detected during a wellness visit, 8% were detected incidentally, and 88% were detected after the owner reported clinical signs suggestive of cancer. Liquid biopsy detected disease in 54.7% (95% confidence interval [CI], 49.5%-59.8%) of patients, including 32% of dogs with early-stage cancer, 48% of preclinical dogs, and 84% of dogs with advanced-stage disease. CONCLUSIONS/CLINICAL IMPORTANCE: Most cases of cancer were diagnosed after the onset of clinical signs; only 4% of dogs had cancer detected using the current standard of care (i.e., wellness visit). Liquid biopsy has the potential to increase detection of cancer when added to a dog's wellness visit.

Clinical experience with next-generation sequencing-based liquid biopsy testing for cancer detection in dogs: a review of 1,500 consecutive clinical cases.

OBJECTIVE: To review ordering patterns, positivity rates, and outcome data for a subset of consecutive samples submitted for a commercially available, blood-based multicancer early-detection liquid biopsy test for dogs using next-generation sequencing at 1 laboratory. SAMPLE: 1,500 consecutively submitted blood samples from client-owned dogs with and without clinical suspicion and/or history of cancer for prospective liquid biopsy testing between December 28, 2021, and June 28, 2022. PROCEDURES: We performed a retrospective observational study, reviewing data from 1,500 consecutive clinical samples submitted for liquid biopsy testing. Outcome data were obtained via medical record review, direct communication with the referring clinic, and/or a patient outcome survey through October 16, 2022. RESULTS: Sixty-four percent (910/1,419) of reportable samples were submitted for cancer screening, 26% (366/1,419) for aid in diagnosis, and 10% (143/1,419) for other indications. The positivity rate was 25.4% (93/366) in aid-in-diagnosis patients and 4.5% (41/910) in screening patients. Outcome data were available for 33% (465/1,401) of patients, and outcomes were classifiable for 428 patients. The relative observed sensitivity was 61.5% (67/109) and specificity was 97.5% (311/319). The positive predictive value was 75.0% (21/28) for screening patients and 97.7% (43/44) for aid-in-diagnosis patients, and the time to diagnostic resolution following a positive result was < 2 weeks in most cases. CLINICAL RELEVANCE: Liquid biopsy using next-generation sequencing represents a novel tool for noninvasive detection of cancer in dogs. Real-world clinical performance meets or exceeds expectations established in the test's clinical validation study.

Detection of Age-Related Somatic Alterations in Canine Blood Using Next-Generation Sequencing-Based Liquid Biopsy: An Analysis of over 4800 Dogs.

Age-related somatic genomic alterations in hematopoietic cell lines have been well characterized in humans; however, this phenomenon has not been well studied in other species. Next-generation sequencing-based liquid biopsy testing for cancer detection was recently developed for dogs and has been used to study the genomic profiles of blood samples from thousands of canine patients since 2021. In this study, 4870 client-owned dogs with and without a diagnosis or suspicion of cancer underwent liquid biopsy testing by this method. Copy number variants detected exclusively in genomic DNA derived from white blood cells (WBC gDNA-specific CNVs) were observed in 126 dogs (2.6%; 95% CI: 2.2-3.1); these copy number variants were absent from matched plasma cell-free DNA, and from tumor tissue in dogs with concurrent cancer. These findings were more common in older dogs and were persistent in WBC gDNA in over 70% of patients, with little to no change in the amplitude of the signal across longitudinal samples. Many of these alterations were observed at recurrent locations in the genome across subjects; the most common finding was a partial loss on CFA25, typically accompanied by a partial gain on the same chromosome. These early findings suggest that age-related somatic alterations may be present at an appreciable frequency in the general canine population. Further research is needed to determine the clinical significance of these findings.

Identifying the activities of physiotherapy practitioners through primary and secondary models of care provided in New South Wales emergency departments.

BACKGROUND: Following the introduction of the emergency department (ED) primary contact physiotherapy role, emergency physiotherapy models of care have evolved and are increasingly being adopted in the Australian ED. This has occurred due to growing ED patient demand and a need for greater workforce flexibility. Since introduction, there here has been limited evaluation of the scope of work physiotherapists are providing in Australian ED. OBJECTIVES: To identify the activities of ED physiotherapists provided through different models of care in NSW. METHODS: Prospective observation study in 19 participating EDs conducted over 6 months between September 2014 and April 2015. RESULTS: The study identified different models of care across participating hospitals where physiotherapists worked independently or in conjunction with a team through a referral service. The individual's scope of work was determined by organisational policy, culture, individual competence, knowledge and skills, and varied significantly between sites. CONCLUSIONS: These findings could guide both ED work flow and the development of multidisciplinary workforce structures to improve the utilisation of the physiotherapy service in EDs. This will allow for better service levels in hospitals, better access for patients and better use of resources.

Molecular characterization and expression analysis of the channel catfish cathepsin D genes.

Cathepsin D is a lysosomal aspartic proteinase that participates in various degradation functions of the cell. In this study, we characterized the cathepsin D genes in channel catfish and found two genes encoding catfish cathepsin D, referred to as cathepsin D1 and D2 genes. These two genes are highly similar in genomic structure and organization, sharing a moderate level of amino acid sequence similarity (56%). Genomic Southern analysis suggested the presence of a single copy of each of the cathepsin D1 and D2 genes. Phylogenetic analysis provided strong evidence that two cathepsin D genes are present in most of the teleost lineage, with cathepsin D2 likely having been lost in some higher vertebrate lineages. The catfish cathepsin D1 and D2 genes are expressed in virtually all the 11 tested tissues (brain, gill, heart, head kidney, trunk kidney, intestine, liver, muscle, skin, spleen, and stomach) on the transcript level, but appear to exhibit greater levels of expression in immune-related tissues and organs. Upon infection with Edwardsiella ictaluri, the expression of the catfish cathepsin D genes showed the most significant changes in liver and head kidney, with time points and magnitude of transcript changes varying between the two genes. We additionally examined bacterially-mediated changes of expression in gill, intestine, and trunk kidney. The fact that bacterial infection can induce expression of the cathepsin D genes and that they appeared to be expressed naturally at higher levels in immune-related organs may suggest that they are an important component of the innate immune response of catfish against bacterial infections.

Impact of the primary contact physiotherapy practitioner role on emergency department care for patients with musculoskeletal injuries in New South Wales.

OBJECTIVES: To determine the impact of the emergency physiotherapy service provided through different models of care on service quality indicators, patient flow, staff and patient satisfaction. METHOD: A mixed method prospective observation study was conducted between September 2014 and April 2015 in 19 EDs where a physiotherapy service is provided. RESULTS: Patients seen by the primary contact physiotherapist (PCP) were associated with a significant reduction in ED length of stay by 108 min, wait time to treatment by 10 min (n = 4 EDs) and time-to-first analgesia by 18 min (n = 19 EDs) compared to those seen through usual care processes. Patients who received care by a doctor first and then physiotherapist (secondary contact model) had a prolonged length of stay compared to other care pathways. High levels of satisfaction with the PCP role were expressed by ED staff (n = 17 EDs) and patients (n = 19 EDs). More than 95% of patients who received care by PCP were satisfied with the management of their condition, understood the advice and discharge information provided and had enough time to ask questions. CONCLUSION: ED implementation of the PCP model may improve patient flow and efficiency of clinical skill utilisation in a complex, high demand workplace.

Identification and relative abundances of mRNA for a gene encoding the vWD domain and three Kazal-type domains in the ovary of giant freshwater prawns, Macrobrachium rosenbergii.

Understanding Macrobrachium rosenbergii ovarian maturation control at the genome level is an important aspect for increasing larvae production. In this study, an ovarian maturation related gene, M. rosenbergii vWD domain and three Kazal-type domains of a gene (MrvWD-Kazal) have been studied. The MrvWD-Kazal gene was isolated using a rapid amplification of cDNA end (RACE) method and the relative abundances of MrvWD-Kazal mRNA in the ovary, hepatopancreas, stomach, intestine and gill were determined by using the quantitative PCR technique. The MrvWD-Kazal gene is composed of 2194 bp with an open reading frame (ORF) of 1998 bp encoding 665 amino acids and has great similarity to the M. nipponense vWD-Kazal gene (91%). The qPCR analyses indicated the relative abundance of MrvWD-Kazal mRNA transcript varied among different stages of ovarian function (P < 0.05), but there were no differences abundance in hepatopancreas, stomach, intestine and gill (P> 0.05). In the ovary, relative abundance of MrvWD-Kazal mRNA transcript gradually increased with ovarian maturation from Stages 1 (Spent; 1.00-fold), to 2 (Proliferative; 3.47-fold) to 3 (Premature; 6.18-fold) and decreased at Stage 4 (Mature; 1.31-fold). Differential relative abundances of MrvWD-Kazal mRNA transcript in the ovary indicate the MrvWD-Kazal protein may have an important function in ovarian maturation of M. rosenbergii. The results of this study also indicate the MrvWD-Kazal is not involved in regulation of the reproductive related function of the hepatopancreas, digestive system (stomach and intestine) and respiratory system (gill).

Effect of foot morphology on center-of-pressure excursion during barefoot walking.

BACKGROUND: The path of the center of pressure during walking varies among individuals by deviating to a greater or lesser extent toward the medial or lateral border of the foot. It is unclear whether this variance is systematic and is affected by foot posture. The aim of this study was to explore the relationship between foot morphology and center-of-pressure excursion during barefoot walking. METHODS: Pressure data were collected from 83 participants whose foot type had been classified as supinated, normal, or pronated according to the Foot Posture Index. Three center-of-pressure variables were analyzed: medial excursion area, lateral excursion area, and total excursion area. RESULTS: Across the spectrum of foot types, we found that the more supinated a participant's foot posture, the larger the area of lateral center-of-pressure excursion, and, conversely, the more pronated the foot posture, the smaller the area of lateral center-of-pressure excursion. Furthermore, the supinated foot type had a relatively larger center-of-pressure total excursion area, and the pronated foot type had a relatively smaller center-of-pressure total excursion area. CONCLUSIONS: These results indicate the importance of assessing foot posture when measuring center of pressure and may help explain regional differences in pain and injury location among foot types.

Development of a Single-Chain Peptide Agonist of the Relaxin-3 Receptor Using Hydrocarbon Stapling.

Structure-activity studies of the insulin superfamily member, relaxin-3, have shown that its G protein-coupled receptor (RXFP3) binding site is contained within its central B-chain α-helix and this helical structure is essential for receptor activation. We sought to develop a single B-chain mimetic that retained agonist activity. This was achieved by use of solid phase peptide synthesis together with on-resin ruthenium-catalyzed ring closure metathesis of a pair of judiciously placed i,i+4 α-methyl, α-alkenyl amino acids. The resulting hydrocarbon stapled peptide was shown by solution NMR spectroscopy to mimic the native helical conformation of relaxin-3 and to possess potent RXFP3 receptor binding and activation. Alternative stapling procedures were unsuccessful, highlighting the critical need to carefully consider both the peptide sequence and stapling methodology for optimal outcomes. Our result is the first successful minimization of an insulin-like peptide to a single-chain α-helical peptide agonist which will facilitate study of the function of relaxin-3.

p,p'-Dichlorodiphenyltrichloroethane (p,p'-DDT) and p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) repress prostate specific antigen levels in human prostate cancer cell lines.

Despite stringent restrictions on their use by many countries since the 1970s, the endocrine disrupting chemicals, DDT and DDE are still ubiquitous in the environment. However, little attention has been directed to p,p'-DDT and the anti-androgen, p,p'-DDE on androgen receptor (AR) target gene transcription in human cells. Inhibitors of androgenic activity may have a deleterious clinical outcome in prostate cancer screens and progression, therefore we determined whether environmentally relevant concentrations of p,p'-DDT and p,p'-DDE negatively impact AR-regulated expression of prostate-specific antigen (PSA), and other AR target genes in human LNCaP and VCaP prostate cancer cells. Quantitative real-time PCR and immuno-blotting techniques were used to measure intracellular PSA, PSMA and AR mRNA and protein levels. We have shown for the first time that p,p'-DDT and p,p'-DDE repressed R1881-inducible PSA mRNA and protein levels in a dose-dependent manner. Additionally, we used the fully automated COBAS PSA detection system to determine that extracellular PSA levels were also significantly repressed. These chemicals achieve this by blocking the recruitment of AR to the PSA promoter region at 10 µM, as demonstrated by the chromatin immunoprecipitation (ChIP) in LNCaP cells. Both p,p'-DDT and p,p'-DDE repressed R1881-inducible AR protein accumulation at 10 µM. Thus, we conclude that men who have been exposed to either DDT or DDE may produce a false-negative PSA test when screening for prostate cancer, resulting in an inaccurate clinical diagnosis. More importantly, prolonged exposure to these anti-androgens may mimic androgen ablation therapy in individuals with prostate cancer, thus exacerbating the condition by inadvertently forcing adaptation to this stress early in the disease.

Second-generation genetic linkage map of catfish and its integration with the BAC-based physical map.

Construction of high-density genetic linkage maps is crucially important for quantitative trait loci (QTL) studies, and they are more useful when integrated with physical maps. Such integrated maps are valuable genome resources for fine mapping of QTL, comparative genomics, and accurate and efficient whole-genome assembly. Previously, we established both linkage maps and a physical map for channel catfish, Ictalurus punctatus, the dominant aquaculture species in the United States. Here we added 2030 BAC end sequence (BES)-derived microsatellites from 1481 physical map contigs, as well as markers from singleton BES, ESTs, anonymous microsatellites, and SNPs, to construct a second-generation linkage map. Average marker density across the 29 linkage groups reached 1.4 cM/marker. The increased marker density highlighted variations in recombination rates within and among catfish chromosomes. This work effectively anchored 44.8% of the catfish BAC physical map contigs, covering ~52.8% of the genome. The genome size was estimated to be 2546 cM on the linkage map, and the calculated physical distance per centimorgan was 393 Kb. This integrated map should enable comparative studies with teleost model species as well as provide a framework for ordering and assembling whole-genome scaffolds.