Asparagine Synthetase Marks a Distinct Dependency Threshold for Cardiomyocyte Dedifferentiation.

BACKGROUND: Adult mammalian cardiomyocytes have limited proliferative capacity, but in specifically induced contexts they traverse through cell-cycle reentry, offering the potential for heart regeneration. Endogenous cardiomyocyte proliferation is preceded by cardiomyocyte dedifferentiation (CMDD), wherein adult cardiomyocytes revert to a less matured state that is distinct from the classical myocardial fetal stress gene response associated with heart failure. However, very little is known about CMDD as a defined cardiomyocyte cell state in transition. METHODS: Here, we leveraged 2 models of in vitro cultured adult mouse cardiomyocytes and in vivo adeno-associated virus serotype 9 cardiomyocyte-targeted delivery of reprogramming factors (Oct4, Sox2, Klf4, and Myc) in adult mice to study CMDD. We profiled their transcriptomes using RNA sequencing, in combination with multiple published data sets, with the aim of identifying a common denominator for tracking CMDD. RESULTS: RNA sequencing and integrated analysis identified Asparagine Synthetase (Asns) as a unique molecular marker gene well correlated with CMDD, required for increased asparagine and also for distinct fluxes in other amino acids. Although Asns overexpression in Oct4, Sox2, Klf4, and Myc cardiomyocytes augmented hallmarks of CMDD, Asns deficiency led to defective regeneration in the neonatal mouse myocardial infarction model, increased cell death of cultured adult cardiomyocytes, and reduced cell cycle in Oct4, Sox2, Klf4, and Myc cardiomyocytes, at least in part through disrupting the mammalian target of rapamycin complex 1 pathway. CONCLUSIONS: We discovered a novel gene Asns as both a molecular marker and an essential mediator, marking a distinct threshold that appears in common for at least 4 models of CMDD, and revealing an Asns/mammalian target of rapamycin complex 1 axis dependency for dedifferentiating cardiomyocytes. Further study will be needed to extrapolate and assess its relevance to other cell state transitions as well as in heart regeneration.

Cellaca® PLX image cytometer as an alternative for immunophenotyping, GFP/RFP transfection efficiencies, and apoptosis analysis.

Cell and gene therapy is a fast-growing field for cancer therapeutics requiring reliable instrumentation and technologies. Key parameters essential for satisfying Chemistry Manufacturing and Controls criteria standards are routinely performed using flow cytometry. Recently, image cytometry was developed for cell characterization and cell-based assays but had not yet demonstrated sufficient sensitivity for surface marker detection. We developed the Cellaca® PLX image cytometry system and the respective methodologies required for immunophenotyping, GFP and RFP transfection/transduction efficiencies, and cell health analyses for routine cell characterization. All samples tested were compared directly to results from the CytoFLEX flow cytometer. PBMCs were stained with T-cell surface markers for immunophenotyping, and results show highly comparable CD3, CD4, and CD8 populations (within 5 %). GFP- or RFP-expressing cell lines were analyzed for transfection/transduction efficiencies, and the percentage positive cells and respective viabilities were equivalent on both systems. Staurosporine-treated Jurkat cells were stained for apoptotic markers, where annexin V and caspase-3 positive cells were within 5 % comparing both instruments. The proposed system may provide a complementary tool for performing routine cell-based experiments with improved efficiency and sensitivity compared to prior image cytometers, which may be significantly valuable to the cell and gene therapy field.

Evolving applications of liquid biopsies in gastrointestinal cancers.

Liquid biopsy is a test that allows for the diagnosis and analysis of cancer by sampling cancer cells or byproducts present in biological fluids such as blood or urine. It has the potential to create a new paradigm in oncologic care, being a less invasive approach than conventional tissue biopsy. Liquid biopsy has multifaceted applications for longitudinal disease monitoring in terms of surveillance, treatment response, and identification of emerging resistance mechanisms. Multiple assays currently exist or are in development for detecting circulating tumor cells, DNA, RNA, exosomes, proteins, fragmentomic markers, and metabolomes. Here, we review the applications of liquid biopsy in gastrointestinal cancers, emphasizing its use in both perioperative and advanced settings. We also examine its role in screening, diagnostics, and other cancer-related scenarios.

Case of metastatic clear cell odontogenic carcinoma with response to chemoimmunotherapy.

Our patient initially presented with 6 months of left jaw pain and gingival bleeding, leading to the discovery of a radiolucent left maxillary mass on dental evaluation. A biopsy confirmed clear cell odontogenic carcinoma, and the patient was treated with definitive surgery and radiation for localised disease. Unfortunately, the patient was found to have pulmonary metastases 3 months after initial management and was subsequently treated with a combination of cytotoxic chemotherapy and immunotherapy with a partial response. To our knowledge, this is the first case demonstrating the successful use of chemoimmunotherapy in metastatic clear cell odontogenic carcinoma.

A cross-sectional study of optometrists' attitudes towards dry eye disease management in Hong Kong: A web-based survey in Hong Kong.

Purpose: This study aimed to explore the practices of optometrists in Hong Kong towards diagnosing and managing dry eye disease (DED). Methods: From September 2021 to March 2022, an online questionnaire was distributed to optometrists in Hong Kong through several professional associations. The questionnaire included questions about the importance and usefulness of various diagnostic tests, as well as inquiries about management strategies and recommended follow-up schedules for DED. Responses were compared between optometrists who were more or less proactive in continuing education to identify potential differences. Results: The analysis included 68 valid responses. Sixty-one of them were Part 1 optometrists that represents 5.5 % of registered Part 1 optometrists back in 2022. Assessment of clinical symptoms was the most commonly performed investigation (93 %) and considered the most important (75 %) procedure in DED assessments, followed by corneal staining and fluorescein tear break-up time. Traditional diagnostic tests were preferred over newer methods, such as osmolarity, which were not yet commonly used. Unpreserved lubricants (90 %) and lid hygiene (63 %) were the primary treatments recommended for mild DED. Optometrists who had more experience and frequent participation in continuing education were more confident in diagnosing and managing DED, and more likely to recommend omega-3 supplements for moderate DED. Conclusion: The diagnostic and management strategies of optometrists in Hong Kong were generally consistent with the recommendations of the Dry Eye Workshop II report. However, standardized DED questionnaires and newer diagnostic tools were not commonly used. Evidence-based optometric care for dry eye management should be encouraged in Hong Kong optometric practice.

Imaging of the hepatic arterial infusion pump: Primer for radiologists.

Hepatic arterial infusion (HAI) pumps are used to deliver liver-directed therapy by allowing the administration of selective chemotherapy to the liver via a catheter implanted most commonly into the gastroduodenal artery connected to a subcutaneous pump. This selective administration helps maximize the chemotherapeutic effect within the hepatic tumors while minimizing systemic toxicity. While HAI therapy has primarily been used to treat liver-only metastatic colorectal cancer, the indications have expanded to other malignancies, including intrahepatic cholangiocarcinoma. Radiologists play an important role in pre-operative planning, assessment of treatment response, and evaluation for potential complications using various imaging studies, including computed tomography angiography, magnetic resonance imaging, and perfusion scintigraphy. This article describes the radiologist's role as part of a multi-disciplinary oncology team to help maximize the success of HAI therapy and also helps radiologists familiarize themselves with various aspects of HAI pumps.

Ocular surface parameters repeatability and agreement -A comparison between Keratograph 5M and IDRA.

PURPOSE: To evaluate the repeatability and agreement in dry eye measurements using Oculus Keratograph 5M (K5M) and SBM Sistemi IDRA (IDRA). METHODS: A total of 108 participants were enrolled and 108 eyes were evaluated. Tear meniscus height (TMH) and first and average non-invasive break-up time (NIBUT) were measured using the K5M and IDRA (order randomly assigned). TMH was measured using the built-in caliper tool while NIBUT was computed by the automatic algorithm of the instruments. RESULTS: The Bland Altman plots analysis showed a good agreement between the two instruments for TMH (95 % Limits of Agreement (LoA), -0.17 to 0.16), but not the first NIBUT (95 % LoA, -8.13 to 14.79) and average NIBUT (95 % LoA, -7.89 to 10.32). The values of the first and average NIBUT measured using IDRA were significantly shorter than in K5M (difference = median (IQR) -2.75 (-6.48- -0.28)s, p < 0.001 and difference = median (IQR) -1.65 (-3.97-1.89)s, p = 0.008 respectively). The TMH (p = 0.037) and NIBUT average (p = 0.033) measured by K5M, as well as the TMH (p = 0.040) measured by IDRA, exhibited unstable measurements across the three measurement times. The remaining parameters exhibited stability with three repeated measurements. CONCLUSION: The NIBUT measurements are not interchangeable between IDRA and K5M, while the TMH was little difference between the two instruments. It is important to exercise caution when using different ocular surface analyzers to minimize errors in comparing multiple measurements.

Multiplexed volumetric CLEM enabled by scFvs provides insights into the cytology of cerebellar cortex.

Mapping neuronal networks is a central focus in neuroscience. While volume electron microscopy (vEM) can reveal the fine structure of neuronal networks (connectomics), it does not provide molecular information to identify cell types or functions. We developed an approach that uses fluorescent single-chain variable fragments (scFvs) to perform multiplexed detergent-free immunolabeling and volumetric-correlated-light-and-electron-microscopy on the same sample. We generated eight fluorescent scFvs targeting brain markers. Six fluorescent probes were imaged in the cerebellum of a female mouse, using confocal microscopy with spectral unmixing, followed by vEM of the same sample. The results provide excellent ultrastructure superimposed with multiple fluorescence channels. Using this approach, we documented a poorly described cell type, two types of mossy fiber terminals, and the subcellular localization of one type of ion channel. Because scFvs can be derived from existing monoclonal antibodies, hundreds of such probes can be generated to enable molecular overlays for connectomic studies.

A petavoxel fragment of human cerebral cortex reconstructed at nanoscale resolution.

To fully understand how the human brain works, knowledge of its structure at high resolution is needed. Presented here is a computationally intensive reconstruction of the ultrastructure of a cubic millimeter of human temporal cortex that was surgically removed to gain access to an underlying epileptic focus. It contains about 57,000 cells, about 230 millimeters of blood vessels, and about 150 million synapses and comprises 1.4 petabytes. Our analysis showed that glia outnumber neurons 2:1, oligodendrocytes were the most common cell, deep layer excitatory neurons could be classified on the basis of dendritic orientation, and among thousands of weak connections to each neuron, there exist rare powerful axonal inputs of up to 50 synapses. Further studies using this resource may bring valuable insights into the mysteries of the human brain.