Uncovering newly identified aldehyde dehydrogenase 2 genetic variants that lead to acetaldehyde accumulation after an alcohol challenge.

BACKGROUND: Aldehyde dehydrogenase 2 (ALDH2) is critical for alcohol metabolism by converting acetaldehyde to acetic acid. In East Asian descendants, an inactive genetic variant in ALDH2, rs671, triggers an alcohol flushing response due to acetaldehyde accumulation. As alcohol flushing is not exclusive to those of East Asian descent, we questioned whether additional ALDH2 genetic variants can drive facial flushing and inefficient acetaldehyde metabolism using human testing and biochemical assays. METHODS: After IRB approval, human subjects were given an alcohol challenge (0.25 g/kg) while quantifying acetaldehyde levels and the physiological response (heart rate and skin temperature) to alcohol. Further, by employing biochemical techniques including human purified ALDH2 proteins and transiently transfected NIH 3T3 cells, we characterized two newly identified ALDH2 variants for ALDH2 enzymatic activity, ALDH2 dimer/tetramer formation, and reactive oxygen species production after alcohol treatment. RESULTS: Humans heterozygous for rs747096195 (R101G) or rs190764869 (R114W) had facial flushing and a 2-fold increase in acetaldehyde levels, while rs671 (E504K) had facial flushing and a 6-fold increase in acetaldehyde levels relative to wild type ALDH2 carriers. In vitro studies with recombinant R101G and R114W ALDH2 enzyme showed a reduced efficiency in acetaldehyde metabolism that is unique when compared to E504K or wild-type ALDH2. The effect is caused by a lack of functional dimer/tetramer formation for R101G and decreased Vmax for both R101G and R114W. Transiently transfected NIH-3T3 cells with R101G and R114W also had a reduced enzymatic activity by ~ 50% relative to transfected wild-type ALDH2 and when subjected to alcohol, the R101G and R114W variants had a 2-3-fold increase in reactive oxygen species formation with respect to wild type ALDH2. CONCLUSIONS: We identified two additional ALDH2 variants in humans causing facial flushing and acetaldehyde accumulation after alcohol consumption. As alcohol use is associated with a several-fold higher risk for esophageal cancer for the E504K variant, the methodology developed here to characterize ALDH2 genetic variant response to alcohol can lead the way precision medicine strategies to further understand the interplay of alcohol consumption, ALDH2 genetics, and cancer.

Accessible interview practices for disabled scientists and engineers.

Increasing representation of people with disabilities in science and engineering will require systemic changes to the culture around support and accommodations. Equitable interview practices can help foster such changes. We, an interdisciplinary group of disabled and nondisabled early-career scientists who care deeply about making science more accessible to all, present a framework of suggestions based on Universal Design principles for improving the accessibility and equitability of interviews for people with disabilities and other underrepresented groups. We discuss potential challenges that may arise when implementing these suggestions and provide questions to guide discussions about addressing them.

Verification of a simplified aneurysm dimensionless flow parameter to predict intracranial aneurysm rupture status.

OBJECTIVES: Aneurysm number (An) is a novel prediction tool utilizing parameters of pulsatility index (PI) and aneurysm geometry. An has been shown to have the potential to differentiate intracranial aneurysm (IA) rupture status. The objective of this study is to investigate the feasibility and accuracy of An for IA rupture status prediction using Australian based clinical data. METHODS: A retrospective study was conducted across three tertiary referral hospitals between November 2017 and November 2020 and all saccular IAs with known rupture status were included. Two sets of An values were calculated based on two sets of PI values previously reported in the literature. RESULTS: Five hundred and four IA cases were included in this study. The results demonstrated no significant difference between ruptured and unruptured status when using An ≥1 as the discriminator. Further analysis showed no strong correlation between An and IA subtypes. The area under the curve (AUC) indicated poor performance in predicting rupture status (AUC1 = 0.55 and AUC2 = 0.56). CONCLUSIONS: This study does not support An ≥1 as a reliable parameter to predict the rupture status of IAs based on a retrospective cohort. Although the concept of An is supported by hemodynamic aneurysm theory, further research is needed before it can be applied in the clinical setting. ADVANCES IN KNOWLEDGE: This study demonstrates that the novel prediction tool, An, proposed in 2020 is not reliable and that further research of this hemodynamic model is needed before it can be incorporated into the prediction of IA rupture status.

Persistent bleeding following biopsy of an epidermoid cyst within the parotid gland.

Epidermoid cysts are benign lesions most commonly found in the skin but which can arise in many other locations including, very rarely the salivary glands. This rarity often leaves them off standard differential lists and can create a diagnostic dilemma. A patient with an incidentally detected parotid mass on MRI underwent core biopsy, which was unfortunately complicated by formation of a pseudoaneurysm and persistent arterial bleeding requiring coil embolisation. The histology showed only keratinous material and, in retrospect, the signal characteristics of the mass were entirely typical of an epidermoid cyst. Recognition of this common, benign entity in a very rare location can obviate the need for invasive tests and potential complications and direct management to more appropriate imaging follow-up.

A novel sorting method for the enrichment of early human spermatocytes from clinical biopsies.

OBJECTIVE: To determine if early spermatocytes can be enriched from a human testis biopsy using fluorescence-activated cell sorting (FACS). DESIGN: Potential surface markers for early spermatocytes were identified using bioinformatics analysis of single-cell RNA-sequenced human testis tissue. Testicular sperm extraction samples from three participants with normal spermatogenesis were digested into single-cell suspensions and cryopreserved. Two to four million cells were obtained from each and sorted by FACS as separate biologic replicates using antibodies for the identified surface markers. A portion from each biopsy remained unsorted to serve as controls. The sorted cells were then characterized for enrichment of early spermatocytes. SETTING: A laboratory study. PATIENTS: Three men with a diagnosis of obstructive azoospermia (age range, 30-40 years). INTERVENTION: None. MAIN OUTCOME MEASURES: Sorted cells were characterized for RNA expression of markers encompassing the stages of spermatogenesis. Sorting markers were validated by their reactivity on human testis formalin-fixed paraffin-embedded tissue. RESULTS: Serine protease 50 (TSP50) and SWI5-dependent homologous recombination repair protein 1 were identified as potential surface proteins specific for early spermatocytes. After FACS sorting, the TSP50-sorted populations accounted for 1.6%-8.9% of total populations and exhibited the greatest average-fold increases in RNA expression for the premeiotic marker stimulated by retinoic acid (STRA8), by 23-fold. Immunohistochemistry showed the staining pattern for TSP50 to be strong in premeiotic undifferentiated embryonic cell transcription factor 1-/doublesex and Mab-3 related transcription factor 1-/STRA8+ spermatogonia as well as SYCP3+/protamine 2- spermatocytes. CONCLUSION: This work shows that TSP50 can be used to enrich early STRA8-expressing spermatocytes from human testicular biopsies, providing a means for targeted single-cell RNA sequencing analysis and in vitro functional interrogation of germ cells during the onset of meiosis. This could enable investigation into details of the regulatory pathways underlying this critical stage of spermatogenesis, previously difficult to enrich from whole tissue samples.

Primary small intestine angiosarcoma mimicking Crohn's disease.

A man in his 40s presented to an emergency department after experiencing worsening abdominal pain for 2 days. Contrast-enhanced CT of the abdomen and pelvis revealed circumferential mural thickening and luminal narrowing of the distal ileum and upstream dilatation of the small intestine, indicating small intestine obstruction. This prompted emergency laparotomy, where two lesions in the distal ileum were identified as the source of his bowel obstruction and resected. Immunohistochemistry of the resected segment revealed a primary small intestine angiosarcoma acting positively for vascular markers ERG and CD31. A subsequent positron emission tomography (PET) scan revealed positive mediastinal metastatic lymphadenopathy without organ metastases.Following his surgery, the patient recovered well and was promptly referred to an oncology unit at a specialised health centre for further treatment. Primary small intestine angiosarcoma is a rare entity in which patients present with non-specific symptoms requiring prompt tissue diagnosis to facilitate multidisciplinary management.

Shot-noise limited optical hybrid based on fused fiber couplers.

We describe a fiber-based coherent receiver topology which utilizes intrinsic phase shifts from fiber couplers to enable instantaneous quadrature projection with shot-noise limited signal-to-noise ratio (SNR). Fused 3 × 3 fiber couplers generate three phase-shifted signals simultaneously that can be combined with quadrature projection methods to detect magnitude and phase unambiguously. We present a novel, to the best of our knowledge, differential detection topology which utilizes a combination of 3 × 3 and 2 × 2 couplers to enable quadrature projection with fully differential detection. We present a mathematical analysis of this 3 × 3 differential detection topology, extended methods for signal calibration, and SNR analysis. We characterize the SNR advantage of this approach and demonstrate a sample application illustrating simultaneous magnitude and phase imaging of a chrome-on-glass test chart.

Translation of non-canonical open reading frames as a cancer cell survival mechanism in childhood medulloblastoma.

A hallmark of high-risk childhood medulloblastoma is the dysregulation of RNA translation. Currently, it is unknown whether medulloblastoma dysregulates the translation of putatively oncogenic non-canonical open reading frames (ORFs). To address this question, we performed ribosome profiling of 32 medulloblastoma tissues and cell lines and observed widespread non-canonical ORF translation. We then developed a stepwise approach using multiple CRISPR-Cas9 screens to elucidate non-canonical ORFs and putative microproteins implicated in medulloblastoma cell survival. We determined that multiple lncRNA-ORFs and upstream ORFs (uORFs) exhibited selective functionality independent of main coding sequences. A microprotein encoded by one of these ORFs, ASNSD1-uORF or ASDURF, was upregulated, associated with MYC-family oncogenes, and promoted medulloblastoma cell survival through engagement with the prefoldin-like chaperone complex. Our findings underscore the fundamental importance of non-canonical ORF translation in medulloblastoma and provide a rationale to include these ORFs in future studies seeking to define new cancer targets.

Parallelized computational 3D video microscopy of freely moving organisms at multiple gigapixels per second.

Wide field of view microscopy that can resolve 3D information at high speed and spatial resolution is highly desirable for studying the behaviour of freely moving model organisms. However, it is challenging to design an optical instrument that optimises all these properties simultaneously. Existing techniques typically require the acquisition of sequential image snapshots to observe large areas or measure 3D information, thus compromising on speed and throughput. Here, we present 3D-RAPID, a computational microscope based on a synchronized array of 54 cameras that can capture high-speed 3D topographic videos over an area of 135 cm2, achieving up to 230 frames per second at spatiotemporal throughputs exceeding 5 gigapixels per second. 3D-RAPID employs a 3D reconstruction algorithm that, for each synchronized snapshot, fuses all 54 images into a composite that includes a co-registered 3D height map. The self-supervised 3D reconstruction algorithm trains a neural network to map raw photometric images to 3D topography using stereo overlap redundancy and ray-propagation physics as the only supervision mechanism. The resulting reconstruction process is thus robust to generalization errors and scales to arbitrarily long videos from arbitrarily sized camera arrays. We demonstrate the broad applicability of 3D-RAPID with collections of several freely behaving organisms, including ants, fruit flies, and zebrafish larvae.

Conserved chamber-specific polyploidy maintains heart function in Drosophila.

Developmentally programmed polyploidy (whole-genome duplication) of cardiomyocytes is common across evolution. Functions of such polyploidy are essentially unknown. Here, in both Drosophila larvae and human organ donors, we reveal distinct polyploidy levels in cardiac organ chambers. In Drosophila, differential growth and cell cycle signal sensitivity leads the heart chamber to reach a higher ploidy/cell size relative to the aorta chamber. Cardiac ploidy-reduced animals exhibit reduced heart chamber size, stroke volume and cardiac output, and acceleration of circulating hemocytes. These Drosophila phenotypes mimic human cardiomyopathies. Our results identify productive and likely conserved roles for polyploidy in cardiac chambers and suggest that precise ploidy levels sculpt many developing tissues. These findings of productive cardiomyocyte polyploidy impact efforts to block developmental polyploidy to improve heart injury recovery.

Genome-wide pooled CRISPR screening in neurospheres.

Spheroid culture systems have allowed in vitro propagation of cells unable to grow in canonical cell culturing conditions, and may capture cellular contexts that model tumor growth better than current model systems. The insights gleaned from genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening of thousands of cancer cell lines grown in conventional culture conditions illustrate the value of such CRISPR pooled screens. It is clear that similar genome-wide CRISPR screens of three-dimensional spheroid cultures will be important for future biological discovery. Here, we present a protocol for genome-wide CRISPR screening of three-dimensional neurospheres. While many in-depth protocols and discussions have been published for more typical cell lines, few detailed protocols are currently available in the literature for genome-wide screening in spheroidal cell lines. For those who want to screen such cell lines, and particularly neurospheres, we provide a step-by-step description of assay development tests to be performed before screening, as well as for the screen itself. We highlight considerations of variables that make these screens distinct from, or similar to, typical nonspheroid cell lines throughout. Finally, we illustrate typical outcomes of neurosphere genome-wide screens, and how neurosphere screens typically produce slightly more heterogeneous signal distributions than more canonical cancer cell lines. Completion of this entire protocol will take 8-12 weeks from the initial assay development tests to deconvolution of the sequencing data.

Translation of non-canonical open reading frames as a cancer cell survival mechanism in childhood medulloblastoma.

A hallmark of high-risk childhood medulloblastoma is the dysregulation of RNA translation. Currently, it is unknown whether medulloblastoma dysregulates the translation of putatively oncogenic non-canonical open reading frames. To address this question, we performed ribosome profiling of 32 medulloblastoma tissues and cell lines and observed widespread non-canonical ORF translation. We then developed a step-wise approach to employ multiple CRISPR-Cas9 screens to elucidate functional non-canonical ORFs implicated in medulloblastoma cell survival. We determined that multiple lncRNA-ORFs and upstream open reading frames (uORFs) exhibited selective functionality independent of the main coding sequence. One of these, ASNSD1-uORF or ASDURF, was upregulated, associated with the MYC family oncogenes, and was required for medulloblastoma cell survival through engagement with the prefoldin-like chaperone complex. Our findings underscore the fundamental importance of non-canonical ORF translation in medulloblastoma and provide a rationale to include these ORFs in future cancer genomics studies seeking to define new cancer targets.

Multi-modal imaging using a cascaded microscope design.

We present a multi-modal fiber array snapshot technique (M-FAST) based on an array of 96 compact cameras placed behind a primary objective lens and a fiber bundle array. Our technique is capable of large-area, high-resolution, multi-channel video acquisition. The proposed design provides two key improvements to prior cascaded imaging system approaches: a novel optical arrangement that accommodates the use of planar camera arrays, and a new ability to acquire multi-modal image data acquisition. M-FAST is a multi-modal, scalable imaging system that can acquire snapshot dual-channel fluorescence images as well as differential phase contrast measurements over a large 6.59 mm × 9.74 mm field-of-view at 2.2-µm center full-pitch resolution.

Conserved Chamber-Specific Polyploidy Maintains Heart Function in Drosophila.

Developmentally programmed polyploidy (whole-genome-duplication) of cardiomyocytes is common across evolution. Functions of such polyploidy are essentially unknown. Here, we reveal roles for precise polyploidy levels in cardiac tissue. We highlight a conserved asymmetry in polyploidy level between cardiac chambers in Drosophila larvae and humans. In Drosophila , differential Insulin Receptor (InR) sensitivity leads the heart chamber to reach a higher ploidy/cell size relative to the aorta chamber. Cardiac ploidy-reduced animals exhibit reduced heart chamber size, stroke volume, cardiac output, and acceleration of circulating hemocytes. These Drosophila phenotypes mimic systemic human heart failure. Using human donor hearts, we reveal asymmetry in nuclear volume (ploidy) and insulin signaling between the left ventricle and atrium. Our results identify productive and likely conserved roles for polyploidy in cardiac chambers and suggest precise ploidy levels sculpt many developing tissues. These findings of productive cardiomyocyte polyploidy impact efforts to block developmental polyploidy to improve heart injury recovery.

Parallelized computational 3D video microscopy of freely moving organisms at multiple gigapixels per second.

To study the behavior of freely moving model organisms such as zebrafish (Danio rerio) and fruit flies (Drosophila) across multiple spatial scales, it would be ideal to use a light microscope that can resolve 3D information over a wide field of view (FOV) at high speed and high spatial resolution. However, it is challenging to design an optical instrument to achieve all of these properties simultaneously. Existing techniques for large-FOV microscopic imaging and for 3D image measurement typically require many sequential image snapshots, thus compromising speed and throughput. Here, we present 3D-RAPID, a computational microscope based on a synchronized array of 54 cameras that can capture high-speed 3D topographic videos over a 135-cm^2 area, achieving up to 230 frames per second at throughputs exceeding 5 gigapixels (GPs) per second. 3D-RAPID features a 3D reconstruction algorithm that, for each synchronized temporal snapshot, simultaneously fuses all 54 images seamlessly into a globally-consistent composite that includes a coregistered 3D height map. The self-supervised 3D reconstruction algorithm itself trains a spatiotemporally-compressed convolutional neural network (CNN) that maps raw photometric images to 3D topography, using stereo overlap redundancy and ray-propagation physics as the only supervision mechanism. As a result, our end-to-end 3D reconstruction algorithm is robust to generalization errors and scales to arbitrarily long videos from arbitrarily sized camera arrays. The scalable hardware and software design of 3D-RAPID addresses a longstanding problem in the field of behavioral imaging, enabling parallelized 3D observation of large collections of freely moving organisms at high spatiotemporal throughputs, which we demonstrate in ants (Pogonomyrmex barbatus), fruit flies, and zebrafish larvae.

More time to kill: A longer liver stage increases T cell-mediated protection against pre-erythrocytic malaria.

Liver stage (LS) Plasmodia mature in 2-2.5 days in rodents compared to 5-6 days in humans. Plasmodium-specific CD8+ T cell expansion differs across these varied timespans. To mimic the kinetics of CD8+ T cells of human Plasmodium infection, a two-dose challenge mouse model that achieved 4-5 days of LS antigen exposure was developed. In this model, mice were inoculated with a non-protective, low dose of late-arresting, genetically attenuated sporozoites to initiate T cell activation and then re-inoculated 2-3 days later with wild-type sporozoites. Vaccines that partially protected against traditional challenge completely protected against two-dose challenge. During the challenge period, CD8+ T cell frequencies increased in the livers of two-dose challenged mice but not in traditionally challenged mice, further suggesting that this model better recapitulates kinetics of CD8+ T cell expansion in humans during the P. falciparum LS. Vaccine development and antigen discovery efforts may be aided by using the two-dose challenge strategy.

Sustainability in interventional radiology: are we doing enough to save the environment?

BACKGROUND: Healthcare waste contributes substantially to the world's carbon footprint. Our aims are to review the current knowledge of Interventional Radiology (IR) waste generation and ways of reducing waste in practice, to quantify the environmental and financial impact of waste generated and address green initiatives to improve IR waste management. METHODS: A systematic literature search was conducted in July 2022 using the Medline and Embase literature databases. The scope of the search included the field of IR as well as operating theatre literature, where relevant to IR practice. RESULTS: One-hundred articles were reviewed and 68 studies met the inclusion criteria. Greening initiatives include reducing, reusing and recycling waste, as well as strict waste segregation. Interventional radiologists can engage with suppliers to reformulate procedure packs to minimize unnecessary items and packaging. Opened but unused equipment can be prevented if there is better communication within the team and increased staff awareness of wasted equipment cost. Incentives to use soon-to-expire equipment can be offered. Power consumption can be reduced by powering down operating room lights and workstations when not in use, changing to Light Emitting Diode (LED) and motion sensor lightings. Surgical hand wash can be replaced with alcohol-based hand rubs to reduce water usage. Common barriers to improving waste management include the lack of leadership, misconceptions regarding infectious risk, lack of data, concerns about increased workload, negative staff attitudes and resistance to change. Education remains a top priority to engage all staff in sustainable healthcare practices. CONCLUSION: Interventional radiologists have a crucial role to play in improving healthcare sustainability. By implementing small, iterative changes to our practice, financial savings, greater efficiency and improved environmental sustainability can be achieved.

Plasmodium vivax latent liver infection is characterized by persistent hypnozoites, hypnozoite-derived schizonts, and time-dependent efficacy of primaquine.

Plasmodium vivax is a malaria-causing pathogen that establishes a dormant form in the liver (the hypnozoite), which can activate weeks, months, or years after the primary infection to cause a relapse, characterized by secondary blood-stage infection. These asymptomatic and undetectable latent liver infections present a significant obstacle to the goal of global malaria eradication. We use a human liver-chimeric mouse model (FRG huHep) to study P. vivax hypnozoite latency and activation in an in vivo model system. Functional activation of hypnozoites and formation of secondary schizonts is demonstrated by first eliminating primary liver schizonts using a schizont-specific antimalarial tool compound, and then measuring recurrence of secondary liver schizonts in the tissue and an increase in parasite RNA within the liver. We also reveal that, while primaquine does not immediately eliminate hypnozoites from the liver, it arrests developing schizonts and prevents activation of hypnozoites, consistent with its clinical activity in humans. Our findings demonstrate that the FRG huHep model can be used to study the biology of P. vivax infection and latency and assess the activity of anti-relapse drugs.