Mutations in cancer-relevant genes are ubiquitous in histologically normal endometrial tissue.

OBJECTIVE: Endometrial cancer (EndoCA) is the most common gynecologic cancer and incidence and mortality rate continue to increase. Despite well-characterized knowledge of EndoCA-defining mutations, no effective diagnostic or screening tests exist. To lay the foundation for testing development, our study focused on defining the prevalence of somatic mutations present in non-cancerous uterine tissue. METHODS: We obtained ≥8 uterine samplings, including separate endometrial and myometrial layers, from each of 22 women undergoing hysterectomy for non-cancer conditions. We ultra-deep sequenced (>2000× coverage) samples using a 125 cancer-relevant gene panel. RESULTS: All women harbored complex mutation patterns. In total, 308 somatic mutations were identified with mutant allele frequencies ranging up to 96.0%. These encompassed 56 unique mutations from 24 genes. The majority of samples possessed predicted functional cancer mutations but curiously no growth advantage over non-functional mutations was detected. Functional mutations were enriched with increasing patient age (p = 0.045) and BMI (p = 0.0007) and in endometrial versus myometrial layers (68% vs 39%, p = 0.0002). Finally, while the somatic mutation landscape shared similar mutation prevalence in key TCGA-defined EndoCA genes, notably PIK3CA, significant differences were identified, including NOTCH1 (77% vs 10%), PTEN (9% vs 61%), TP53 (0% vs 37%) and CTNNB1 (0% vs 26%). CONCLUSIONS: An important caveat for future liquid biopsy/DNA-based cancer diagnostics is the repertoire of shared and distinct mutation profiles between histologically unremarkable and EndoCA tissues. The lack of selection pressure between functional and non-functional mutations in histologically unremarkable uterine tissue may offer a glimpse into an unrecognized EndoCA protective mechanism.

Deep molecular tracking over the 12-yr development of endometrial cancer from hyperplasia in a single patient.

Although the progressive histologic steps leading to endometrial cancer (EndoCA), the most common female reproductive tract malignancy, from endometrial hyperplasia are well-established, the molecular changes accompanying this malignant transformation in a single patient have never been described. We had the unique opportunity to investigate the paired histologic and molecular features associated with the 12-yr development of EndoCA in a postmenopausal female who could not undergo hysterectomy and instead underwent progesterone treatment. Using a specially designed 58-gene next-generation sequencing panel, we analyzed a total of 10 sequential biopsy samples collected over this time frame. A total of eight pathogenic/likely pathogenic mutations in seven genes, APC, ARID1A, CTNNB1, CDKN2A, KRAS, PTEN, and TP53, were identified. A PTEN nonsense mutation p.W111* was present in all samples analyzed except histologically normal endometrium. Apart from this PTEN mutation, the only other recurrent mutation was KRAS G12D, which was present in six biopsy samplings, including histologically normal tissue obtained at the patient's first visit but not detectable in the cancer. The PTEN p.W111* mutant allele fractions were lowest in benign, inactive endometrial glands (0.7%), highest in adenocarcinoma (36.9%), and, notably, were always markedly reduced following progesterone treatment. To our knowledge, this report provides the first molecular characterization of EndoCA development in a single patient. A single PTEN mutation was present throughout the 12 years of cancer development. Importantly, and with potential significance toward medical and nonsurgical management of EndoCA, progesterone treatments were consistently noted to markedly decrease PTEN mutant allele fractions to precancerous levels.

Native American genetic ancestry and pigmentation allele contributions to skin color in a Caribbean population.

Our interest in the genetic basis of skin color variation between populations led us to seek a Native American population with genetically African admixture but low frequency of European light skin alleles. Analysis of 458 genomes from individuals residing in the Kalinago Territory of the Commonwealth of Dominica showed approximately 55% Native American, 32% African, and 12% European genetic ancestry, the highest Native American genetic ancestry among Caribbean populations to date. Skin pigmentation ranged from 20 to 80 melanin units, averaging 46. Three albino individuals were determined to be homozygous for a causative multi-nucleotide polymorphism OCA2NW273KV contained within a haplotype of African origin; its allele frequency was 0.03 and single allele effect size was -8 melanin units. Derived allele frequencies of SLC24A5A111T and SLC45A2L374F were 0.14 and 0.06, with single allele effect sizes of -6 and -4, respectively. Native American genetic ancestry by itself reduced pigmentation by more than 20 melanin units (range 24-29). The responsible hypopigmenting genetic variants remain to be identified, since none of the published polymorphisms predicted in prior literature to affect skin color in Native Americans caused detectable hypopigmentation in the Kalinago.

The variation in skin colour of modern humans is a product of thousands of years of natural selection. All human ancestry can be traced back to African populations, which were dark-skinned to protect them from the intense UV rays of the sun. Over time, humans spread to other parts of the world, and people in the northern latitudes with lower UV developed lighter skin through natural selection. This was likely driven by a need for vitamin D, which requires UV rays for production. Separate genetic mechanisms were involved in the evolution of lighter skin in each of the two main branches of human migration: the European branch (which includes peoples on the Indian subcontinent and Europe) and the East Asian branch (which includes East Asia and the Americas). A variant of the gene SLC24A5 is the primary contributor to lighter skin colour in the European branch, but a corresponding variant driving light skin colour evolution in the East Asian branch remains to be identified. One obstacle to finding such variants is the high prevalence of European ancestry in most people groups, which makes it difficult to separate the influence of European genes from those of other populations. To overcome this issue, Ang et al. studied a population that had a high proportion of Native American and African ancestors, but a relatively small proportion of European ancestors, the Kalinago people. The Kalinago live on the island of Dominica, one of the last Caribbean islands to be colonised by Europeans. Ang et al. were able to collect hundreds of skin pigmentation measurements and DNA samples of the Kalinago, to trace the effect of Native American ancestry on skin colour. Genetic analysis confirmed their oral history records of primarily Native American (55%) ­ one of the highest of any Caribbean population studied to date ­ compared with African (32%) and European (12%) ancestries. Native American ancestry had the highest effect on pigmentation and reduced it by more than 20 melanin units, while the European mutations in the genes SLC24A5 and SLC45A2 and an African gene variant for albinism only contributed 5, 4 and 8 melanin units, respectively. However, none of the so far published gene candidates responsible for skin lightening in Native Americans caused a detectable effect. Therefore, the gene responsible for lighter skin in Native Americans/East Asians has yet to be identified. The work of Ang et al. represents an important step in deciphering the genetic basis of lighter skin colour in Native Americans or East Asians. A better understanding of the genetics of skin pigmentation may help to identify why, for example, East Asians are less susceptible to melanoma than Europeans, despite both having a lighter skin colour. It may also further acceptance of how variations in human skin tones are the result of human migration, random genetic variation, and natural selection for pigmentation in different solar environments.

The Importance of Local and Global Social Ties for the Mental Health and Well-Being of Recently Resettled Refugee-Background Women in Australia.

Social connections are foundational to the human condition and are inherently disrupted when people are forcibly displaced from their home countries. At a time of record high global forced migration, there is value in better understanding how refugee-background individuals engage theirsocial supports or ties in resettlement contexts. A mixed methods research design aimed to understand the complexities of how 104 refugee-background women experienced their social networks in the first few months of resettlement in Australia. One of the research activities involved participants completing a survey with both quantitative and qualitative components. The quantitative analyses identified the impact of post-migration living difficulties that represented social stressors (worry about family, loneliness and boredom, feeling isolated, and racial discrimination) on the women's mental health outcomes in the months following resettlement. The qualitative data highlighted the complexities of social relationships serving as both stressors and sources of support, and the importance of recognizing extended families and supports around the globe. The findings point to the need for nuanced accounts of the social contexts surrounding refugee resettlement as important influences able to promote trauma-informed and gender sensitive practices to support mental health and well-being in new settings.

Impact of the COVID-19 Pandemic on the Global Delivery of Mental Health Services and Telemental Health: Systematic Review.

BACKGROUND: The COVID-19 pandemic required mental health services around the world to adapt quickly to the new restrictions and regulations put in place to reduce the risk of transmission. As face-to-face contact became difficult, virtual methods were implemented to continue to safely provide mental health care. However, it is unclear to what extent service provision transitioned to telemental health worldwide. OBJECTIVE: We aimed to systematically review the global research literature on how mental health service provision adapted during the first year of the pandemic. METHODS: We searched systematically for quantitative papers focusing on the impact of the COVID-19 pandemic on mental health services published until April 13, 2021, in the PubMed, Embase, medRxiv, and bioXriv electronic bibliographic databases, using the COVID-19 Open Access Project online platform. The screening process and data extraction were independently completed by at least two authors, and any disagreement was resolved by discussion with a senior member of the team. The findings were summarized narratively in the context of each country's COVID-19 Stringency Index, which reflects the stringency of a government's response to COVID-19 restrictions at a specific time. RESULTS: Of the identified 24,339 records, 101 papers were included after the screening process. Reports on general services (n=72) showed that several countries' face-to-face services reduced their activities at the start of the pandemic, with reductions in the total number of delivered visits and with some services forced to close. In contrast, telemental health use rapidly increased in many countries across the world at the beginning of the pandemic (n=55), with almost complete virtualization of general and specialistic care services by the end of the first year. Considering the reported COVID-19 Stringency Index values, the increased use of virtual means seems to correspond to periods when the Stringency Index values were at their highest in several countries. However, due to specific care requirements, telemental health could not be used in certain subgroups of patients, such as those on clozapine or depot treatments and those who continued to need face-to-face visits. CONCLUSIONS: During the pandemic, mental health services had to adapt quickly in the short term, implementing or increasing the use of telemental health services across the globe. Limited access to digital means, poor digital skills, and patients' preferences and individual needs may have contributed to differences in implementing and accessing telemental health services during the pandemic. In the long term, a blended approach, combining in-person and virtual modalities, that takes into consideration the needs, preferences, and digital skills of patients may better support the future development of mental health services. It will be required to improve confidence with digital device use, training, and experience in all modalities for both clinicians and service users.

Medical students' perceptions of general practice as a career; a phenomenological study using socialisation theory.

The ageing population and push to community care has significantly increased the workload of General Practitioners (GPs) in the UK and internationally. In an attempt to tackle this, NHS England has promised 5000 more GPs by 2020/21; however, recruitment is in crisis with GP training posts remaining unfilled. Little research has been carried out to assess the fundamental questions of what medical students' perceptions of General Practice are and what shapes their perceptions at medical school. We aimed to explore medical students' conceptualisations of being a GP and specifically the role of the medical school in shaping their perceptions. Two focus groups of year one and year four medical students were undertaken using an interpretive phenomenological approach. Our study has revealed that medical students perceive General Practice to lack prestige and challenge. These perceptions come, at least in part, from a process of socialisation within medical school, whereby medical students internalise and adopt their role models' perceptions and values, and the values portrayed by the hidden curriculum in their medical school culture. Perceived external pressures to pursue a career in General Practice can have a negative influence and medical schools should be made aware of this.

Effect of choline chloride premedication on xylazine-induced hypoxaemia in sheep.

OBJECTIVE: To determine the anti-inflammatory efficacy of choline in vivo and in vitro and to investigate the anti-inflammatory mechanisms of choline. STUDY DESIGN: Randomized, controlled studies. ANIMALS: In vivo trials used 16 Romney sheep. In vitro experiments utilized RAW 264.7 mouse macrophage cells. METHODS: Hypoxaemia induced in 16 sheep by intravenous (IV) injection of 50 µg kg-1 xylazine, an α-2 agonist, was measured in sheep at 0, 1 and 4 minutes using arterial blood gas analysis with and without 50 mg kg-1 IV choline chloride premedication. Cell culture studies used enzyme-linked immunosorbent assay to measure the release of tumour necrosis factor (TNF-α) from lipopolysaccharide (LPS) stimulated macrophages with and without choline chloride premedication. TNF-α release was compared to thalidomide suppressed and untreated cells. RESULTS: Choline premedication in sheep mitigated a reduction in arterial partial pressure of oxygen (PaO2) but did not prevent development of clinically significant hypoxaemia. Decrease in mean PaO2 of choline treated sheep was 6.36 kPa (47.7 mmHg) compared to 9.81 kPa (73.6 mmHg) in control sheep. In vitro studies demonstrate that choline administered concurrent with LPS activation did not significantly suppress TNF-α expression but that treatment of cells with choline 10 minutes prior to LPS activation did significantly suppress TNF-α expression. Choline pretreated cells expressed 23.99 ± 4.52 ng mg-1 TNF-α while LPS only control cells expressed 33.83 ± 3.20 ng mg-1. CONCLUSIONS: Choline is able to prevent macrophage activation in vitro when administered prior to LPS activation and may reduce hypoxaemia in sheep developing pulmonary oedema after xylazine administration. This effect requires premedication with choline. CLINICAL RELEVANCE: Pharmacological manipulation of autonomic inflammatory responses holds promise for the treatment of inflammation. However, the complex cellular mechanisms involved in this reflex means that an adequate therapy should approach multiple pathways and mechanisms of the inflammatory response.

Unravelling the spatial variation of nitrous oxide emissions from a step-feed plug-flow full scale wastewater treatment plant.

Plug-flow activated sludge reactors (ASR) that are step-feed with wastewater are widely adopted in wastewater treatment plants (WWTPs) due to their ability to maximise the use of the organic carbon in wastewater for denitrification. Nitrous oxide (N2O) emissions are expected to vary along these reactors due to pronounced spatial variations in both biomass and substrate concentrations. However, to date, no detailed studies have characterised the impact of the step-feed configuration on emission variability. Here we report on the results from a comprehensive online N2O monitoring campaign, which used multiple gas collection hoods to simultaneously measure emission along the length of a full-scale, step-fed, plug-flow ASR in Australia. The measured N2O fluxes exhibited strong spatial-temporal variation along the reactor path. The step-feed configuration had a substantial influence on the N2O emissions, where the N2O emission factors in sections following the first and second step feed were 0.68% ± 0.09% and 3.5% ± 0.49% of the nitrogen load applied to each section. The relatively high biomass-specific nitrogen loading rate in the second section of the reactor was most likely cause of the high emissions from this section.

Undifferentiated murine embryonic stem cells used to model the effects of the blue-green algal toxin cylindrospermopsin on preimplantation embryonic cell proliferation.

Undifferentiated mouse embryonic stem cell (mES) proliferation in vitro resembles aspects of in vivo pre-implantation embryonic development. mES were used to assess the embryo-toxicity of cylindrospermopsin (CYN), a water contaminant with an Australian Drinking Water Guideline (ADWG) of 1 µg/L. mES exposed to 0-1 µg/mL CYN for 24-168 h were subjected to an optimised crystal violet viability assay. mES exposed to retinoic acid ± 1 µg/L CYN differentiated into neural-like cells confirmed by morphological examination and RT-PCR for Oct4, Brachyury and Nestin. The CYN No Observed Effect Concentration (OEC) was 0.5 µg/mL, the Lowest OEC was 1 µg/mL (p < 0.001, n = 3), and the IC50 was 0.86 µg/mL after 24 h. The ADWG 1 µg/L CYN did not affect differentiation or proliferation after 72 h, but decreased proliferation after 168 h (p < 0.05). We conclude that higher algal bloom-associated CYN concentrations have the potential to impair in vivo pre-implantation development, and the mES crystal violet assay has broad application to screening environmental toxins.

Evaluation of granular sludge for secondary treatment of saline municipal sewage.

This study examined the impact of chemical oxygen demand (COD) loading and dissolved oxygen (DO) concentration on the stability and performance of granular sludge treating high saline municipal sewage. Under high DO concentrations of 4.0-7.0 mg/L, and COD loading rates of 0.98 and 1.55 kg/m(3)/d, rapid settling granules were established within four weeks of start-up. Under the highest COD load, a reduction in DO lead to the rapid deterioration of the sludge volume index (SVI) and washout of granules due to prolific growth of the filament Thiothrix Type 021N. Conversely, when operated under a lower COD load, a reduction in DO concentration had no adverse impact on the stability of SVI and granules. A decrease in DO also improved nitrogen removal performance, where simultaneous removal of ammonium (98%), total nitrogen (86%) and BOD5 (98%) were achieved when median DO concentrations were between 1.0 and 1.5 mg/L. Phosphate removal was lower than expected, however the level of biological phosphate removal activity observed appeared sufficient to maintain granule stability, even under low DO concentrations. Nitrous oxide emissions were also characterised, which ranged between 2.3 and 6.8% of the total nitrogen load. Our results confirmed that granular sludge is a viable option for the treatment of saline sewage.

Developmental roles of Drosophila tRNA processing endonuclease RNase ZL as revealed with a conditional rescue system.

Drosophila RNase Z(L) (dRNaseZ) belongs to a family of endoribonucleases with a major role in tRNA 3'-end processing. The biochemical function of RNase Z(L) is conserved from yeast to human. Here we present a study of its biological function during Drosophila development. In flies, dRNaseZ provides a non-redundant function, as the RNZ(ED24) knockout (KO) mutation causes early larval lethality. Mosaic and conditional rescue techniques were employed to determine dRNaseZ requirements at later stages. We found that dRNaseZ activity is essential for all phases of fly development that involve cell division, including growth of adult tissue progenitors during larval and metamorphic stages, and gametogenesis in adults. At the cellular level, two major phenotypes were identified-cell growth deficiency in endoreplicating tissues and cell cycle arrest in mitotic tissues. While cell growth and proliferation are both dependant on protein synthesis, the two phenotypes displayed reliance on different dRNaseZ functions. We found that dRNaseZ KO completely blocks tRNA maturation without diminishing the abundance of mature tRNA molecules. Our data indicate that growth arrest of endoreplicating cells is primarily attributed to the relocation of the pool of mature tRNAs into the nuclei causing a decrease in translation efficiency. Mitotically dividing cells appear to be less dependent on translation machinery as they maintain their normal size when deprived of dRNaseZ activity, but rather display a cell cycle arrest at the G2-M transition.

Functional assessment of human coding mutations affecting skin pigmentation using zebrafish.

A major challenge in personalized medicine is the lack of a standard way to define the functional significance of the numerous nonsynonymous, single nucleotide coding variants that are present in each human individual. To begin to address this problem, we have used pigmentation as a model polygenic trait, three common human polymorphisms thought to influence pigmentation, and the zebrafish as a model system. The approach is based on the rescue of embryonic zebrafish mutant phenotypes by "humanized" zebrafish orthologous mRNA. Two hypomorphic polymorphisms, L374F in SLC45A2, and A111T in SLC24A5, have been linked to lighter skin color in Europeans. The phenotypic effect of a second coding polymorphism in SLC45A2, E272K, is unclear. None of these polymorphisms had been tested in the context of a model organism. We have confirmed that zebrafish albino fish are mutant in slc45a2; wild-type slc45a2 mRNA rescued the albino mutant phenotype. Introduction of the L374F polymorphism into albino or the A111T polymorphism into slc24a5 (golden) abolished mRNA rescue of the respective mutant phenotypes, consistent with their known contributions to European skin color. In contrast, the E272K polymorphism had no effect on phenotypic rescue. The experimental conclusion that E272K is unlikely to affect pigmentation is consistent with a lack of correlation between this polymorphism and quantitatively measured skin color in 59 East Asian humans. A survey of mutations causing human oculocutaneous albinism yielded 257 missense mutations, 82% of which are theoretically testable in zebrafish. The developed approach may be extended to other model systems and may potentially contribute to our understanding the functional relationships between DNA sequence variation, human biology, and disease.

Skin color variation in Orang Asli tribes of Peninsular Malaysia.

Pigmentation is a readily scorable and quantitative human phenotype, making it an excellent model for studying multifactorial traits and diseases. Convergent human evolution from the ancestral state, darker skin, towards lighter skin colors involved divergent genetic mechanisms in people of European vs. East Asian ancestry. It is striking that the European mechanisms result in a 10-20-fold increase in skin cancer susceptibility while the East Asian mechanisms do not. Towards the mapping of genes that contribute to East Asian pigmentation there is need for one or more populations that are admixed for ancestral and East Asian ancestry, but with minimal European contribution. This requirement is fulfilled by the Senoi, one of three indigenous tribes of Peninsular Malaysia collectively known as the Orang Asli. The Senoi are thought to be an admixture of the Negrito, an ancestral dark-skinned population representing the second of three Orang Asli tribes, and regional Mongoloid populations of Indo-China such as the Proto-Malay, the third Orang Asli tribe. We have calculated skin reflectance-based melanin indices in 492 Orang Asli, which ranged from 28 (lightest) to 75 (darkest); both extremes were represented in the Senoi. Population averages were 56 for Negrito, 42 for Proto-Malay, and 46 for Senoi. The derived allele frequencies for SLC24A5 and SLC45A2 in the Senoi were 0.04 and 0.02, respectively, consistent with greater South Asian than European admixture. Females and individuals with the A111T mutation had significantly lighter skin (p = 0.001 and 0.0039, respectively). Individuals with these derived alleles were found across the spectrum of skin color, indicating an overriding effect of strong skin lightening alleles of East Asian origin. These results suggest that the Senoi are suitable for mapping East Asian skin color genes.

Exploring the process of adjusting to diabetic kidney disease.

The psychological process of adjusting to diabetic kidney disease (DKD) has been afforded little attention within previous literature. This study aimed to explore the process of adjusting to DKD as a secondary condition to type 1 diabetes. A qualitative method was used in the study design. Eleven participants were interviewed using a semi-structured and exploratory approach. Through inductive thematic analysis, three super-ordinate themes were identified from the data: coming to an understanding over time, managing the impact of DKD and facing the future. The themes demonstrate that the DKD population has complex physical and psychological needs at all stages of their condition. Making sense of kidney disease as a secondary condition and integration of this information with existing beliefs was integral to the adjustment process. Attempts to manage uncertainty and change included asserting control of the situation and use of cognitive strategies. The results support the use of psychological models of adjustment in conceptualising the 'process' of adjustment and suggest the need for psychological interventions to support this population.

The acoustic characteristics of professional opera singers performing in chorus versus solo mode.

In this study, members of a professional opera chorus were recorded using close microphones, while singing in both choral and solo modes. The analysis included computation of long-term average spectra (LTAS) for the two song sections performed and calculation of singing power ratio (SPR) and energy ratio (ER), which provide an indication of the relative energy in the singer's formant region. Vibrato rate and extent were determined from two matched vowels, and SPR and ER were calculated for these vowels. Subjects sung with equal or more power in the singer's formant region in choral versus solo mode in the context of the piece as a whole and in individual vowels. There was no difference in vibrato rate and extent between the two modes. Singing in choral mode, therefore, required the ability to use a similar vocal timbre to that required for solo opera singing.

Representational momentum in scenes: learning spatial layout.

In four experiments, we examined whether watching a scene from the perspective of a camera rotating across it allowed participants to recognize or identify the scene's spatial layout. Completing a representational momentum (RM) task, participants viewed a smoothly animated display and then indicated whether test probes were in the same position as they were in the final view of the animation. We found RM anticipations for the camera's movement across the scene, with larger distortions resulting from camera rotations that brought objects into the viewing frame compared with camera rotations that took objects out of the viewing frame. However, the RM task alone did not lead to successful recognition of the scene's map or identification of spatial relations between objects. Watching a scene from a rotating camera's perspective and making position judgments is not sufficient for learning spatial layout.