Body fat and circulating leptin levels in the captive short-beaked echidna (Tachyglossus aculeatus).

It is possible that the reproductive strategy of the short-beaked echidna is related to seasonal changes in fat deposition and energy availability, regulated by seasonal changes in endocrine function. We predicted that circulating leptin levels would be directly proportional to adiposity during most of the year, but that a change in this relationship would occur during the pre-breeding season to allow increased fat deposition. To test this hypothesis, we made use of a captive colony of echidnas to describe and quantify changes in fat distribution and the adipostatic hormone leptin. First we assessed seasonal changes in circulating leptin levels, body mass and adiposity for three male and three female adult echidnas maintained on a standard diet. Second, we explored the relationship between circulating leptin levels and increased caloric intake for an additional five adult female echidnas that were provided with supplemented nutrition. Third we visualised fat distribution in male and female adult echidnas using magnetic resonance imaging (MRI) before and after the breeding season, to determine where fat is deposited in this species. For echidnas maintained on the standard diet, there were no seasonal changes in body mass, body fat or plasma leptin levels. However, female echidnas provided with supplemented nutrition had significantly elevated plasma leptin levels during the breeding season, compared to the pre-and post- breeding periods. MRI showed substantial subcutaneous fat depots extending dorso-laterally from the base of the skull to the base of the tail, in both sexes. Pre-breeding season, both sexes had considerable fat deposition in the pelvic/rump region, whilst the female echidna accumulated most fat in the abdominal region. This study shows that male and female echidnas accumulate body fat in the pelvic/rump and the abdominal regions, respectively and that circulating leptin may promote fattening in female echidnas during the breeding season by means of leptin resistance. However, further research is required to evaluate the precise relationship between seasonal changes in leptin and adiposity.

The emerging predictive and prognostic role of HER2 in HER2-negative early breast cancer: a retrospective study.

PURPOSE: Many patients with early breast cancer (eBC) undergoing neoadjuvant chemotherapy do not achieve pathological complete response (pCR), which is a prognostic factor. We examined the role of HER2-low expression in predicting pCR and prognosis in HER2-negative eBC. METHODS: We evaluated patients with stage I-III HER2-negative BC, treated between 2013 and 2023 at The Royal Marsden NHS Foundation Trust, London. Tumors were classified based on estrogen receptor (ER) status and into HER2-low and HER2-zero subgroups. We analyzed pCR rates, relapse-free survival (RFS) and overall survival (OS). RESULTS: 754 patients were included in the analysis. pCR rate was 8.9% in the ER+ /HER2-low, 16.5% in the ER+ /HER2-zero, 38.9% in the ER- ER-/HER2-low and 35.9% in the ER-/HER2-zero eBC (p < 0.001). Multivariable analysis showed a significantly lower pCR rate in HER2-low compared to HER2-zero BC in the ER+ subgroup. At a median follow-up of 63.8 months (59.9-67.4), we observed longer OS in HER2-low compared to HER2-zero patients in the overall and in the ER+ population. There was no predictive or prognostic impact of HER2-low status in the ER- population. CONCLUSION: This study supports the interpretation of HER2 status as a possible prognostic and predictive biomarker for HER2-negative eBC, especially among patients with ER+ disease.

CURE on yeast genes of unknown function increases students' bioinformatics proficiency and research confidence.

Course-based undergraduate research experiences (CUREs) can reduce barriers to research opportunities while increasing student knowledge and confidence. However, the number of widely adopted, easily transferable CUREs is relatively small. Here, we describe a CURE aimed at determining the function of poorly characterized Saccharomyces cerevisiae genes. More than 20 years after sequencing of the yeast genome, nearly 10% of open reading frames (ORFs) still have at least one uncharacterized Gene Ontology (GO) term. We refer to these genes as "ORFans" and formed a consortium aimed at assigning functions to them. Specifically, over 70 faculty members attended summer workshops to learn the bioinformatics workflow and basic laboratory techniques described herein. Ultimately, this CURE was adapted for implementation at 34 institutions, resulting in over 1,300 students conducting course-based research on ORFans. Pre-/post-tests confirmed that students gained both (i) an understanding of gene ontology and (ii) knowledge regarding the use of bioinformatics to assign gene function. After using these data to craft their own hypotheses, then testing their predictions by constructing and phenotyping deletion strains, students self-reported significant gains in several areas, including computer modeling and exposure to a project where no one knows the outcome. Interestingly, most net gains self-reported by ORFan Gene Project participants were greater than published findings for CUREs assessed with the same survey instrument. The surprisingly strong impact of this CURE may be due to the incoming lack of experience of ORFan Project participants and/or the independent thought required to develop testable hypotheses from complex data sets.

Impact of dose reductions on adjuvant abemaciclib efficacy for patients with high-risk early breast cancer: analyses from the monarchE study.

In monarchE, adjuvant abemaciclib significantly improved invasive disease-free survival (IDFS) and distant relapse-free survival (DRFS), with sustained benefit beyond the 2-year treatment period. Abemaciclib dose reductions were allowed to proactively manage adverse events. Exploratory analyses to investigate the impact of dose reductions on efficacy were conducted. Across the three patient subgroups as defined by relative dose intensity (≤66%, 66-93%, ≥93%), the estimated 4-year IDFS rates were generally consistent (87.1%, 86.4%, and 83.7%, respectively). In the time-dependent Cox proportional hazard model, the effect of abemaciclib was consistent at the full dose compared to being reduced to a lower dose (IDFS hazard ratio: 0.905; 95% confidence interval: 0.727, 1.125; DRFS hazard ratio: 0.942; 95% confidence interval: 0.742, 1.195). These analyses showed that the efficacy of adjuvant abemaciclib was not compromised by protocol mandated dose reductions for patients with node positive, hormone receptor positive, human epidermal growth factor 2-negative, high-risk early breast cancer.

Role of DNase Activity in Human Sperm DNA Fragmentation.

In this clinical era of intracytoplasmic sperm injection (ICSI), where a single spermatozoon is chosen for fertilization, the diagnostic functionality of the classical parameters typically associated with fertilization, such as sperm concentration, sperm motility, acrosome integrity, and mitochondria, is perhaps becoming less critical. In contrast, the contribution of sperm DNA quality to our understanding of the impact of male fertility within the context of ICSI is gaining increasing interest and importance. Even with respect to natural conception, high levels of sperm DNA fragmentation (SDF) in the ejaculate can adversely affect reproductive outcomes. However, the precise origin of SDF pathology in sperm cells is often ambiguous and most likely to be multifactorial. Hence, the genetic makeup of an individual, unbalanced REDOX processes, enzymatic activity, environmental and lifestyle factors, and even damage during sperm handling in the laboratory all operate in a unique and often synergistic manner to produce or induce sperm DNA damage. Surprisingly, the contribution of active enzymes as potential agents of SDF has received much less attention and, therefore, is likely to be underrated. This review highlights the roles of different enzymes related to the degradation of sperm DNA as possible effectors of DNA molecules in spermatozoa.

Chinook salmon depth distributions on the continental shelf are shaped by interactions between location, season, and individual condition.

BACKGROUND: Ecological and physical conditions vary with depth in aquatic ecosystems, resulting in gradients of habitat suitability. Although variation in vertical distributions among individuals provides evidence of habitat selection, it has been challenging to disentangle how processes at multiple spatio-temporal scales shape behaviour. METHODS: We collected thousands of observations of depth from > 300 acoustically tagged adult Chinook salmon Oncorhynchus tshawytscha, spanning multiple seasons and years. We used these data to parameterize a machine-learning model to disentangle the influence of spatial, temporal, and dynamic oceanographic variables while accounting for differences in individual condition and maturation stage. RESULTS: The top performing machine learning model used bathymetric depth ratio (i.e., individual depth relative to seafloor depth) as a response. We found that bathymetry, season, maturation stage, and spatial location most strongly influenced Chinook salmon depth. Chinook salmon bathymetric depth ratios were deepest in shallow water, during winter, and for immature individuals. We also identified non-linear interactions among covariates, resulting in spatially-varying effects of zooplankton concentration, lunar cycle, temperature and oxygen concentration. CONCLUSIONS: Our results suggest Chinook salmon vertical habitat use is a function of ecological interactions, not physiological constraints. Temporal and spatial variation in depth distributions could be used to guide management decisions intended to reduce fishery impacts on Chinook salmon. More generally, our findings demonstrate how complex interactions among bathymetry, seasonality, location, and life history stage regulate vertical habitat selection.

Activity and safety of enobosarm, a novel, oral, selective androgen receptor modulator, in androgen receptor-positive, oestrogen receptor-positive, and HER2-negative advanced breast cancer (Study G200802): a randomised, open-label, multicentre, multinational, parallel design, phase 2 trial.

BACKGROUND: The androgen receptor is a tumour suppressor in oestrogen receptor-positive breast cancer. The activity and safety of enobosarm, an oral selective androgen receptor modulator, was evaluated in women with oestrogen receptor (ER)-positive, HER2-negative, and androgen receptor (AR)-positive disease. METHODS: Women who were postmenopausal (aged ≥18 years) with previously treated ER-positive, HER2-negative, locally advanced or metastatic breast cancer with an Eastern Cooperative Oncology Group performance status of 0-2 were enrolled in a randomised, open-label, multicentre, multinational, parallel design, phase 2 trial done at 35 cancer treatment centres in nine countries. Participants were stratified on the setting of immediately preceding endocrine therapy and the presence of bone-only metastasis and randomly assigned (1:1) to 9 mg or 18 mg oral enobosarm daily using an interactive web response system. The primary endpoint was clinical benefit rate at 24 weeks in those with centrally confirmed AR-positive disease (ie, the evaluable population). This trial is registered with ClinicalTrials.gov (NCT02463032). FINDINGS: Between Sept 10, 2015, and Nov 28, 2017, 136 (79%) of 172 patients deemed eligible were randomly assigned to 9 mg (n=72) or 18 mg (n=64) oral enobosarm daily. Of these 136 patients, 102 (75%) patients formed the evaluable population (9 mg, n=50; 18 mg, n=52). The median age was 60·5 years (IQR 52·3-69·3) in the 9 mg group and 62·5 years (54·0-69·3) in the 18 mg group. The median follow-up was 7·5 months (IQR 2·9-14·1). At 24 weeks, 16 (32%, 95% CI 20-47) of 50 in the 9 mg group and 15 (29%, 17-43) of 52 in the 18 mg group had clinical benefit. Six (8%) of 75 patients who received 9 mg and ten (16%) of 61 patients who received 18 mg had grade 3 or grade 4 drug-related adverse events, most frequently increased hepatic transaminases (three [4%] of 75 in the 9 mg group and two [3%] of 61 in the 18 mg group), hypercalcaemia (two [3%] and two [3%]), and fatigue (one [1%] and two [3%]). Four deaths (one in the 9 mg group and three in the 18 mg group) were deemed unrelated to the study drug. INTERPRETATION: Enobosarm has anti-tumour activity in patients with ER-positive, HER2-negative advanced breast cancer, showing that AR activation can result in clinical benefit, supporting further clinical investigation of selective AR activation strategies for the treatment of AR-positive, ER-positive, HER2-negative advanced breast cancer. FUNDING: GTx.

Long-term patient-reported outcomes from monarchE: Abemaciclib plus endocrine therapy as adjuvant therapy for HR+, HER2-, node-positive, high-risk, early breast cancer.

BACKGROUND: In monarchE, abemaciclib demonstrated a sustained benefit in invasive disease-free survival and a tolerable safety profile at 42-months median follow-up. With no expected disease-related symptoms, therapies in the adjuvant setting should preserve quality of life (QoL). With all patients off abemaciclib, we report updated patient-reported outcomes (PROs) for the full 2-year treatment period and follow-up. METHODS: Patients completed PROs including FACT-B, FACT-ES, and FACIT-Fatigue at baseline, 3, 6, 12, 18, and 24 months during treatment, and 1, 6, and 12 months after treatment discontinuation. Mixed effects repeated measures model estimated changes from baseline within and between arms for QoL scales and individual items. Meaningful changes were prespecified and no statistical testing was performed. Frequencies of responses to items associated with relevant adverse events and treatment bother were summarized. RESULTS: At baseline, completion rates for PRO instruments were >96 %. Mean changes from baseline for all QoL scales were numerically similar within and between arms (ie, less than prespecified thresholds). The same was observed for all individual items, except diarrhea. Within abemaciclib arm, meaningful differences for diarrhea were observed at 3 and 6 months (mean increases of 1.19 and 1.03 points on 5-point scale, respectively). During treatment, most patients in both arms (69-78 %) reported being bothered "a little bit" or "not at all" by side effects. Overall, patterns for fatigue were similar between arms. During post-treatment follow-up, PROs in both arms were similar to baseline. CONCLUSION: PRO findings confirm a tolerable and reversible toxicity profile for abemaciclib. QoL was preserved with the addition of adjuvant abemaciclib to endocrine therapy, supporting its use in patients with HR+, HER2-, high-risk early breast cancer.

Adjuvant Abemaciclib Plus Endocrine Therapy for Hormone Receptor-Positive, Human Epidermal Growth Factor Receptor 2-Negative, High-Risk Early Breast Cancer: Results From a Preplanned monarchE Overall Survival Interim Analysis, Including 5-Year Efficacy Outcomes.

Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned co-primary or secondary analyses are not yet available. Clinical trial updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported.Two years of adjuvant abemaciclib combined with endocrine therapy (ET) resulted in a significant improvement in invasive disease-free survival (IDFS) and distant relapse-free survival (DRFS) that persisted beyond the 2-year treatment period in patients with hormone receptor-positive, human epidermal growth factor receptor 2-negative, node-positive, high-risk early breast cancer (EBC). Here, we report 5-year efficacy results from a prespecified overall survival (OS) interim analysis. In the intent-to-treat population, with a median follow-up of 54 months, the benefit of abemaciclib was sustained with hazard ratios of 0.680 (95% CI, 0.599 to 0.772) for IDFS and 0.675 (95% CI, 0.588 to 0.774) for DRFS. This persistence of abemaciclib benefit translated to continuous separation of the curves with a deepening in 5-year absolute improvement in IDFS and DRFS rates of 7.6% and 6.7%, respectively, compared with rates of 6% and 5.3% at 4 years and 4.8% and 4.1% at 3 years. With fewer deaths in the abemaciclib plus ET arm compared with the ET-alone arm (208 v 234), statistical significance was not reached for OS. No new safety signals were observed. In conclusion, abemaciclib plus ET continued to reduce the risk of developing invasive and distant disease recurrence beyond the completion of treatment. The increasing absolute improvement at 5 years is consistent with a carryover effect and further supports the use of abemaciclib in patients with high-risk EBC.

Immunohistochemical characterization of the immune cell response during chlamydial infection in the male and female koala (Phascolarctos cinereus) reproductive tract.

Chlamydiosis is one of the main causes of the progressive decline of koala populations in eastern Australia. While histologic, immunologic, and molecular studies have provided insights into the basic function of the koala immune system, the in situ immune cell signatures during chlamydial infection of the reproductive tract in koalas have not been investigated. Thirty-two female koalas and 47 males presented to wildlife hospitals with clinical signs suggestive of Chlamydia infection were euthanized with the entire reproductive tract collected for histology; immunohistochemistry (IHC) for T-cell (CD3ε, CD4, and CD8α), B-cell (CD79b), and human leukocyte antigen (HLA)-DR markers; and quantitative real-time polymerase chain reaction (rtPCR) for Chlamydia pecorum. T-cells, B-cells, and HLA-DR-positive cells were observed in both the lower and upper reproductive tracts of male and female koalas with a statistically significant associations between the degree of the inflammatory reaction; the number of CD3, CD4, CD79b, and HLA-DR positive cells; and the PCR load. CD4-positive cells were negatively associated with the severity of the gross lesions. The distribution of immune cells was also variable according to the location within the genital tract in both male and female koalas. These preliminary results represent a step forward towards further exploring mechanisms behind chlamydial infection immunopathogenesis, thus providing valuable information about the immune response and infectious diseases in free-ranging koalas.

The relationship between sperm nuclear DNA fragmentation, mitochondrial DNA fragmentation, and copy number in normal and abnormal human ejaculates.

BACKGROUND: While it is common to clinically evaluate sperm nuclear DNA fragmentation, less attention has been given to sperm mitochondrial DNA. Recently, a digital PCR assay has allowed accurate estimation of the proportion of fragmented mtDNA molecules and relative copy number. OBJECTIVES: To determine the correlation of classical sperm parameters, average mtDNA copies per spermatozoon and the level of mtDNA fragmentation (SDF-mtDNA) to that of nuclear DNA fragmentation (SDF-nDNA), measured as the proportion of global, single-strand DNA (SDF-SSBs) and double-strand DNA breaks (SDF-DSBs). To determine whether the level of nuclear and mitochondrial DNA fragmentation and/or copy number can differentiate normozoospermic from non-normozoospermic samples. MATERIALS AND METHODS: Ejaculates from 29 normozoospermic and 43 non-normozoospermic were evaluated. SDF was determined using the sperm chromatin dispersion assay. mtDNA copy number and SDF-mtDNA were analyzed using digital PCR assays. RESULTS: Relative mtDNA copy increased as sperm concentration or motility decreased, or abnormal morphology increased. Unlike SDF-mtDNA, mtDNA copy number was not correlated with SDF-nDNA. SDF-mtDNA increased as the concentration or proportion of non-vital sperm increased; the higher the mtDNA copy number, the lower the level of fragmentation. Non-normozoospermic samples showed double the level of SDF-nDNA compared to normozoospermic (median 25.00 vs. 13.67). mtDNA copy number per spermatozoon was 3× higher in non-normozoospermic ejaculates (median 16.06 vs. 4.99). Although logistic regression revealed SDF-Global and mtDNA copy number as independent risk factors for non-normozoospermia, when SDF-Global and mtDNA copy number were combined, ROC curve analysis resulted in an even stronger discriminatory ability for predicting the probability of non-normozoospermia (AUC = 0.85, 95% CI 0.76-0.94, p < 0.001). CONCLUSION: High-quality ejaculates show lower nuclear SDF and retain less mtDNA copies, with approximately half of them fragmented, so that the absolute number of non-fragmented mtDNA molecules per spermatozoon is extremely low.

An evaluation of DNA sample source and molecular markers to determine gender in the short-beaked echidna (Tachyglossus aculeatus).

The short-beaked echidna is sexually monomorphic such that gender identification without veterinary intervention is challenging. The aim of this study was to evaluate and compare the most optimal noninvasive genetic source by extracting echidna genomic DNA (gDNA) from fecal scats, plucked hair, and quills to perform genetic sex testing using a range of molecular markers. Sex determination of 14 captive short-beaked echidnas was determined by amplifying isolated DNA from noninvasive samples, targeting two Y chromosome (male-specific) genes (mediator complex subunit 26 Y-gametologue [CRSPY] and anti-Müllerian hormone Y-gametologue [AMHY]), in addition to four confirmed sex-specific RADseq markers. Results of noninvasive samples were compared with blood samples and clinical records. Receiver operating characteristic curves were used to assess accuracy of sex determination of markers for each sample type. The gender of the echidnas was successfully identified on 75% of occasions using fecal samples, 90.6% occasions using hair, and 84.6% occasions with quills. Overall, the male-specific RADseq markers accurately identified the sex of echidnas with all sample types for 90% of animals; compared with 81.5% using CRSPY, and 82.0% using AMHY to identify sex. Collection of hair, quills, and feces provides a useful alternative to invasively collected samples, however, the accuracy of results depends on sample type and genetic marker selected. We found gender determination in the short-beaked echidna was most accurate using four male-specific RADseq markers on gDNA isolated from blood and hair. The noninvasive genetic sexing techniques documented here will inform and facilitate husbandry and genetic management of captive echidna populations.

Design of a randomized, placebo-controlled study evaluating efficacy and safety of a cancer preventative vaccine in dogs.

Preventative anti-cancer vaccination strategies have long been hampered by the challenge of targeting the diverse array of potential tumor antigens, with successes to date limited to cancers with viral etiologies. Identification and vaccination against frameshift neoantigens conserved across multiple species and tumor histologies is a potential cancer preventative strategy currently being investigated. Companion dogs spontaneously develop cancers at a similar incidence to those in people and are a complementary comparative patient population for the development of novel anti-cancer therapeutics. In addition to an intact immune system with tumors that arise in an autochthonous tumor microenvironment, dogs also have a shorter lifespan and temporally compressed tumor natural history as compared to humans, which allows for more rapid evaluation of safety, immunogenicity, and efficacy of cancer vaccination strategies. Here we describe the study protocol for the Vaccination Against Canine Cancer Study (VACCS), the largest interventional cancer clinical trial conducted in companion dogs to date. In addition to safety and immunogenicity, the primary endpoint of VACCS is the cumulative incidence (CI) of dogs developing malignant neoplasia of any type at the end of the study period. Secondary endpoints include changes in incidence of specific tumor types, survival times following neoplasia diagnosis, and all-cause mortality.

Increasing Incremental Burden of Surgical Bleeding Associated with Multiple Comorbidities as Measured by the Elixhauser Comorbidity Index: A Retrospective Database Analysis.

Purpose: Disruptive bleeding can complicate surgical procedures, increasing resource use, and impacting patients' well-being. This study aims to elucidate the impact of comorbidity on the risk of disruptive surgical-related bleeding and selected transfusion-associated complications, as well as the incremental cost of such bleeding. Patients and Methods: This retrospective analysis of the Premier Healthcare Database included patients who were age ≥18 years and who had a procedure of interest between 1-Jan-2019-31-Dec-2019: cholecystectomy, coronary artery bypass grafting, cystectomy, hepatectomy, hysterectomy, pancreatectomy, peripheral vascular, thoracic, and valve procedures (first=index). The Elixhauser comorbidity index was assessed on index date and patients were grouped by cumulative comorbidity score (0, 1, 2, 3, 4, 5, ≥6). Outcomes, all measured as in-hospital during index, included bleeding (diagnosis and/or intervention for bleeding), transfusion-associated complications (diagnosis of infection, acute renal failure, or vascular events), and incremental total hospital costs associated with bleeding. Multivariable generalized linear models were used to examine the association of comorbidity/bleeding with outcomes. Results: Of the 304,074 patients included, 7% experienced bleeding. The Elixhauser scores were distributed as follows: 0=29%, 1=23%, 2=18%, 3=12%, 4=8%, 5=5%, ≥6=5%. Odds of bleeding significantly increased with Elixhauser score: 1 comorbidity vs 0 (odds ratio [OR] =1.30, 95% confidence interval [95% CI] =1.19-1.43), and this trend continued to surge (≥6 comorbidities [OR=3.22, 95% CI=2.94-3.53]). Similarly, the odds of transfusion-associated complications significantly increased with comorbidities score: 1 comorbidity vs 0 (OR=2.14, 95% CI=1.88-2.34), ≥6 comorbidities vs 0 (OR=12.37, 95% CI=10.80-14.16). The incremental cost of bleeding also increased with comorbidities score; per-patient costs with and without bleeding were $18,132 vs $13,190, p < 0.001 among patients with 0 comorbidities and $28,952 vs $19,623, p < 0.001 among patients with ≥6 comorbidities. Conclusion: Higher comorbidity burden was associated with significant increases in the risk of surgical bleeding, subsequent transfusion-related complications, and incremental cost burden of bleeding.

Gut microbiota in the short-beaked echidna (Tachyglossus Aculeatus) shows stability across gestation.

Indigenous gut microbial communities (microbiota) play critical roles in health and may be especially important for the mother and fetus during pregnancy. Monotremes, such as the short-beaked echidna, have evolved to lay and incubate an egg, which hatches in their pouch where the young feeds. Since both feces and eggs pass through the cloaca, the fecal microbiota of female echidnas provides an opportunity for vertical transmission of microbes to their offspring. Here, we characterize the gut/fecal microbiome of female short-beaked echidnas and gain a better understanding of the changes that may occur in their microbiome as they go through pregnancy. Fecal samples from four female and five male echidnas were obtained from the Currumbin Wildlife Sanctuary in Queensland and sequenced to evaluate bacterial community structure. We identified 25 core bacteria, most of which were present in male and female samples. Genera such as Fusobacterium, Bacteroides, Escherichia-Shigella, and Lactobacillus were consistently abundant, regardless of sex or gestation stage, accounting for 58.00% and 56.14% of reads in male and female samples, respectively. The echidna microbiome remained stable across the different gestation stages, though there was a significant difference in microbiota composition between male and female echidnas. This study is the first to describe the microbiome composition of short-beaked echidnas across reproductive phases and allows the opportunity for this novel information to be used as a metric of health to aid in the detection of diseases triggered by microbiota dysbiosis.

Anatomy of the Female Koala Reproductive Tract.

The koala (Phascolarctos cinereus), while being an iconic Australian marsupial, has recently been listed as endangered. To establish an improved understanding of normal reproductive anatomy, this paper brings together unpublished research which has approached the topic from two perspectives: (1) the establishment of an artificial insemination program, and (2) the definition of Chlamydia spp.-derived histopathological changes of the female koala urogenital system. Based on the presentation and histological processing of over 70 opportunistic specimens, recovered from wildlife hospitals in Southeast Queensland (Australia), we describe the gross and microanatomy of the koala ovary, oviduct, uteri, vaginal complex, and urogenital sinus during the interestrous, proliferative, and luteal phases of the reproductive cycle.

Addendum to: The relationship between sperm nuclear DNA fragmentation, mitochondrial DNA fragmentation and copy number in normal and abnormal human ejaculates.

BACKGROUND: While the kinetics of human sperm nuclear DNA fragmentation (SDF-nDNA) following ejaculation have been described, the dynamics and relationships of mitochondrial DNA copy number per spermatozoon (mtDNAcn) and fragmentation (SDF-mtDNA) remain unexplored. OBJECTIVES: To compare post-ejaculatory kinetics of mtDNAcn, SDF-mtDNA and SDF-nDNA, global, single-strand DNA breaks (SDF-SSBs) and double-strand DNA breaks (SDF-DSBs) in normozoospermic and non-normozoospermic samples. MATERIALS AND METHODS: 28 normozoospermic and 43 non-normozoospermic ejaculates were evaluated at 0, 6, 24 and 48 h of incubation in vitro. SDF-nDNA was determined by sperm chromatin dispersion (SCD) assays. mtDNAcn and SDF-mtDNA were analysed by dPCR. RESULTS: SDF-nDNA-global values increased as a consequence of quadratic SDF-SSBs and linear SDF-DSBs kinetics. Non-normozoospermic samples showed a slower SDF-global rate between 6-24 h, due to lesser SSBs production. Regarding SDF-DSBs, non-normozoospermic samples exhibited a faster initial increase rate, followed by a slower final increment. The mtDNAcn median value decreased linearly, being 3.2× higher in non-normozoospermics at all time points; mtDNAcn in both cohorts reduced to half of the baseline by 48 h. mtDNAcn was identified as a risk factor for discriminating non-normozoospermia, a finding that was further strengthen when combined with SDF-Global or SDF-DSBs values. SDF-mtDNA frequencies were identical, increasing over time correspondingly in both cohorts. The mtDNA fragmentation rate was initially fast, decreasing progressively with time for both cohorts; half of the initially unfragmented copies were fragmented after 48 h. Rates of mtDNAcn loss and SDF-mtDNA increase were only marginally correlated with the rates of nuclear fragmentation. CONCLUSION: mtDNA fragmentation and loss occur post ejaculation. Their dynamics are likely to be associated with different and/or uncoupled mechanisms to that which cause nuclear DNA fragmentation. Our results indicate that while mtDNA fragmentation is not influenced by the sperm quality, the number of copies of sperm mtDNAcn can potentially serve as a risk factor for predicting non-normozoospermia.

Landscape of baseline and acquired genomic alterations in circulating tumor DNA with abemaciclib alone or with endocrine therapy in advanced breast cancer.

PURPOSE: To identify potential predictors of response and resistance mechanisms in patients with hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer (ABC) treated with the CDK4/6 inhibitor abemaciclib +/- endocrine therapy (ET), baseline and acquired genomic alterations in circulating tumor DNA (ctDNA) were analyzed and associated with clinical outcomes. PATIENTS AND METHODS: MONARCH 3: postmenopausal women with HR+, HER2- ABC and no prior systemic therapy in the advanced setting were randomized to abemaciclib or placebo plus nonsteroidal aromatase inhibitor (NSAI). nextMONARCH: women with HR+, HER2- metastatic breast cancer that progressed on/after prior ET and chemotherapy were randomized to abemaciclib alone (two doses) or plus tamoxifen. Baseline and end-of-treatment plasma samples from patients in MONARCH 3 and nextMONARCH (monotherapy arms) were analyzed to identify somatic genomic alterations. Association between genomic alterations and median progression-free survival (mPFS) was assessed. RESULTS: Most patients had ≥1 genomic alteration detected in baseline ctDNA. In MONARCH 3, abemaciclib+NSAI was associated with improved mPFS versus placebo+NSAI, regardless of baseline alterations. ESR1 alterations were less frequently acquired in the abemaciclib+NSAI arm than placebo+NSAI. Acquired alterations potentially associated with resistance to abemaciclib +/- NSAI included RB1 and MYC. CONCLUSIONS: In MONARCH 3, certain baseline ctDNA genomic alterations were prognostic for ET but not predictive of abemaciclib response. Further studies are warranted to assess whether ctDNA alterations acquired during abemaciclib treatment differ from other CDK4/6 inhibitors. Findings are hypothesis-generating, further exploration is warranted into mechanisms of resistance to abemaciclib and ET.

The koala (Phascolarctos cinereus) prostate: a comprehensive histological and immunohistochemical investigation.

The prostate of the koala (Phascolarctos cinereus), and of marsupials more generally, is the primary contributor of seminal fluid, yet comparatively little is known about its microanatomy or biochemistry. This study explored evidence of parenchymal segmentation of the koala prostate. The prostate of three sexually mature koalas were processed for histopathology, histochemistry (Masson's trichrome, Alcian Blue, periodic acid Schiff staining), and immunohistochemistry using basal (tumor protein 63, cytokeratin 14) and luminal (cytokeratin 8/18, prostate specific antigen, androgen receptor) markers. Results confirmed clear segmentation of the koala prostate into three zones, anterior, central, and posterior, characterized by differences in the proportion of glandular tissue, as well as the thickness of collagen fibers; there were also distinct differences in the secretions produced in each zone. Based on immunohistochemistry, the koala prostate showed evidence of both basal proliferative and luminal secretory cells. The ratio of cell types varied across the three segments, with the central segment housing the highest density of basal cells. Globular bodies produced in the anterior zone were shown to possess the same markers as those described for human prostasomes. This study is the first to comprehensively document the marsupial prostate in terms of microanatomy and corresponding immunohistochemistry. While further biochemical analysis, such as proteomics of each segment will better define the relative functions of each tissue, the data presented here are consistent with the hypothesis that the koala prostate potentially represents an example of an ontological stage in the evolutionary differentiation of male eutherian accessory glands.

Clinical and Economic Burden Associated with Disruptive Surgical Bleeding: A Retrospective Database Analysis.

Background: Hemostatic agents are used to control surgical bleeding; however, some patients experience disruptive bleeding despite the use of hemostats. In patients receiving hemostats, we compared clinical and economic outcomes between patients with vs without disruptive bleeding during a variety of surgical procedures. Methods: This was a retrospective analysis of the Premier Healthcare Database. Study patients were age ≥18 with a hospital encounter for one of 9 procedures with evidence of hemostatic agent use between 1-Jan-2019 and 31-Dec-2019: cholecystectomy, coronary artery bypass grafting (CABG), cystectomy, hepatectomy, hysterectomy, pancreatectomy, peripheral vascular, thoracic, and valve procedures (first procedure = index). Patients were grouped by presence vs absence of disruptive bleeding. Outcomes evaluated during index included intensive care unit (ICU) admission/duration, ventilator use, operating room time, length of stay (LOS), in-hospital mortality, and total hospital costs; 90-day all-cause inpatient readmission was also evaluated. Multivariable analyses were used to examine the association of disruptive bleeding with outcomes, adjusting for patient, procedure, and hospital/provider characteristics. Results: The study included 51,448 patients; 16% had disruptive bleeding (range 1.5% for cholecystectomy to 44.4% for valve). In procedures for which ICU and ventilator use is not routine, disruptive bleeding was associated with significant increases in the risks of admission to ICU and requirement for ventilator (all p≤0.05). Across all procedures, disruptive bleeding was also associated with significant incremental increases in days spent in ICU (all p≤0.05, except CABG), LOS (all p≤0.05, except thoracic), and total hospital costs (all p≤0.05); 90-day all-cause inpatient readmission, in-hospital mortality, and operating room time were higher in the presence of disruptive bleeding and varied in statistical significance across procedures. Conclusion: Disruptive bleeding was associated with substantial clinical and economic burden across a wide variety of surgical procedures. Findings emphasize the need for more effective and timely intervention for surgical bleeding events.