Cationic Microbubbles for Non-Selective Binding of Cavitation Nuclei to Bacterial Biofilms.

The presence of multi-drug resistant biofilms in chronic, persistent infections is a major barrier to successful clinical outcomes of therapy. The production of an extracellular matrix is a characteristic of the biofilm phenotype, intrinsically linked to antimicrobial tolerance. The heterogeneity of the extracellular matrix makes it highly dynamic, with substantial differences in composition between biofilms, even in the same species. This variability poses a major challenge in targeting drug delivery systems to biofilms, as there are few elements both suitably conserved and widely expressed across multiple species. However, the presence of extracellular DNA within the extracellular matrix is ubiquitous across species, which alongside bacterial cell components, gives the biofilm its net negative charge. This research aims to develop a means of targeting biofilms to enhance drug delivery by developing a cationic gas-filled microbubble that non-selectively targets the negatively charged biofilm. Cationic and uncharged microbubbles loaded with different gases were formulated and tested to determine their stability, ability to bind to negatively charged artificial substrates, binding strength, and, subsequently, their ability to adhere to biofilms. It was shown that compared to their uncharged counterparts, cationic microbubbles facilitated a significant increase in the number of microbubbles that could both bind and sustain their interaction with biofilms. This work is the first to demonstrate the utility of charged microbubbles for the non-selective targeting of bacterial biofilms, which could be used to significantly enhance stimuli-mediated drug delivery to the bacterial biofilm.

Cryo-plasma FIB/SEM volume imaging of biological specimens.

Serial focussed ion beam scanning electron microscopy (FIB/SEM) enables imaging and assessment of subcellular structures on the mesoscale (10 nm to 10 µm). When applied to vitrified samples, serial FIB/SEM is also a means to target specific structures in cells and tissues while maintaining constituents' hydration shells for in situ structural biology downstream. However, the application of serial FIB/SEM imaging of non-stained cryogenic biological samples is limited due to low contrast, curtaining, and charging artefacts. We address these challenges using a cryogenic plasma FIB/SEM. We evaluated the choice of plasma ion source and imaging regimes to produce high-quality SEM images of a range of different biological samples. Using an automated workflow we produced three-dimensional volumes of bacteria, human cells, and tissue, and calculated estimates for their resolution, typically achieving 20-50 nm. Additionally, a tag-free localisation tool for regions of interest is needed to drive the application of in situ structural biology towards tissue. The combination of serial FIB/SEM with plasma-based ion sources promises a framework for targeting specific features in bulk-frozen samples (>100 µm) to produce lamellae for cryogenic electron tomography.

Ot2Rec: A semi-automatic, extensible, multi-software tomographic reconstruction workflow.

Electron cryo-tomography is an imaging technique for probing 3D structures with at the nanometer scale. This technique has been used extensively in the biomedical field to study the complex structures of proteins and other macromolecules. With the advancement in technology, microscopes are currently capable of producing images amounting to terabytes of data per day, posing great challenges for scientists as the speed of processing of the images cannot keep up with the ever-higher throughput of the microscopes. Therefore, automation is an essential and natural pathway on which image processing-from individual micrographs to full tomograms-is developing. In this paper, we present Ot2Rec, an open-source pipelining tool which aims to enable scientists to build their own processing workflows in a flexible and automatic manner. The basic building blocks of Ot2Rec are plugins which follow a unified application programming interface structure, making it simple for scientists to contribute to Ot2Rec by adding features which are not already available. In this paper, we also present three case studies of image processing using Ot2Rec, through which we demonstrate the speedup of using a semi-automatic workflow over a manual one, the possibility of writing and using custom (prototype) plugins, and the flexibility of Ot2Rec which enables the mix-and-match of plugins. We also demonstrate, in the Supplementary Material, a built-in reporting feature in Ot2Rec which aggregates the metadata from all process being run, and output them in the Jupyter Notebook and/or HTML formats for quick review of image processing quality. Ot2Rec can be found at https://github.com/rosalindfranklininstitute/ot2rec.

Okapi-EM: A napari plugin for processing and analyzing cryogenic serial focused ion beam/scanning electron microscopy images.

An emergent volume electron microscopy technique called cryogenic serial plasma focused ion beam milling scanning electron microscopy (pFIB/SEM) can decipher complex biological structures by building a three-dimensional picture of biological samples at mesoscale resolution. This is achieved by collecting consecutive SEM images after successive rounds of FIB milling that expose a new surface after each milling step. Due to instrumental limitations, some image processing is necessary before 3D visualization and analysis of the data is possible. SEM images are affected by noise, drift, and charging effects, that can make precise 3D reconstruction of biological features difficult. This article presents Okapi-EM, an open-source napari plugin developed to process and analyze cryogenic serial pFIB/SEM images. Okapi-EM enables automated image registration of slices, evaluation of image quality metrics specific to pFIB-SEM imaging, and mitigation of charging artifacts. Implementation of Okapi-EM within the napari framework ensures that the tools are both user- and developer-friendly, through provision of a graphical user interface and access to Python programming.

Multi-level determinants of breast cancer screening among Malay-Muslim women in Singapore: a sequential mixed-methods study.

OBJECTIVE: Ethnic disparity persists despite equal access to health care in Singapore, with Malay-Muslim women having the lowest mammogram uptake rate and highest breast cancer mortality rate. We sought to understand barriers to and facilitators for mammogram uptake in this community. METHODS: We used a sequential mixed-methods design to first explore reasons for screening and not screening for breast cancer, then determine factors associated with screening and regular screening in a survey. We used maximum variation sampling for semi-structured in-depth interviews to select screeners and non-screeners of diverse ages and educational levels. Twenty-three Malay-Muslim women aged 40-69 years old were interviewed. Themes were categorized using thematic analysis. For the survey, we applied the Health Belief Model, Social Ecological Model, as well as themes from the interviews and findings from previous studies on factors influencing screening in Muslim women to guide questionnaire design. We surveyed 271 Malay-Muslim women aged 50-69 years old in a nationally representative sample. Multivariable logistic regression was used to determine factors associated with ever gone for mammogram and regular mammogram uptake. RESULTS: Through in-depth-interviews, we found perceived benefits of saving lives and breasts from early detection, reminders from doctors and husbands, symptoms, perceived test from God, and personal responsibility to care for one's health facilitated screening. Barriers were perceived low susceptibility, inconvenience, cost, negative psychological effects, misinformation on mammogram triggering cancer cells, religious beliefs, perceived negative outcomes from mammography and distrust of doctor. From the survey, we found cues from health care professionals and needing symptoms before deciding to go for mammogram to be significantly associated with ever gone for mammogram and regular mammogram. Factors associated with ever gone for mammogram only included age, perceived benefits of saving lives from early detection, perceived importance of mammogram, Punishing Allah Reappraisal, and modesty. Factors associated with regular mammogram only included household income, perceived structural barriers to screening and perceived susceptibility to breast cancer. CONCLUSIONS: Mammogram uptake is affected by multiple levels of influence. Interventions to promote screening should be designed with multiple stakeholders including doctors, religious leaders and women who had attended screening.

Skin fibroblast metabolomic profiling reveals that lipid dysfunction predicts the severity of Friedreich's ataxia.

Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by a triplet guanine-adenine-adenine (GAA) repeat expansion in intron 1 of the FXN gene, which leads to decreased levels of the frataxin protein. Frataxin is involved in the formation of iron-sulfur (Fe-S) cluster prosthetic groups for various metabolic enzymes. To provide a better understanding of the metabolic status of patients with FRDA, here we used patient-derived fibroblast cells as a surrogate tissue for metabolic and lipidomic profiling by liquid chromatography-high resolution mass spectrometry. We found elevated HMG-CoA and ß-hydroxybutyrate-CoA levels, implying dysregulated fatty acid oxidation, which was further demonstrated by elevated acyl-carnitine levels. Lipidomic profiling identified dysregulated levels of several lipid classes in FRDA fibroblast cells when compared with non-FRDA fibroblast cells. For example, levels of several ceramides were significantly increased in FRDA fibroblast cells; these results positively correlated with the GAA repeat length and negatively correlated with the frataxin protein levels. Furthermore, stable isotope tracing experiments indicated increased ceramide synthesis, especially for long-chain fatty acid-ceramides, in FRDA fibroblast cells compared with ceramide synthesis in healthy control fibroblast cells. In addition, PUFA-containing triglycerides and phosphatidylglycerols were enriched in FRDA fibroblast cells and negatively correlated with frataxin levels, suggesting lipid remodeling as a result of FXN deficiency. Altogether, we demonstrate patient-derived fibroblast cells exhibited dysregulated metabolic capabilities, and their lipid dysfunction predicted the severity of FRDA, making them a useful surrogate to study the metabolic status in FRDA.

SuRVoS 2: Accelerating Annotation and Segmentation for Large Volumetric Bioimage Workflows Across Modalities and Scales.

As sample preparation and imaging techniques have expanded and improved to include a variety of options for larger sized and numbers of samples, the bottleneck in volumetric imaging is now data analysis. Annotation and segmentation are both common, yet difficult, data analysis tasks which are required to bring meaning to the volumetric data. The SuRVoS application has been updated and redesigned to provide access to both manual and machine learning-based segmentation and annotation techniques, including support for crowd sourced data. Combining adjacent, similar voxels (supervoxels) provides a mechanism for speeding up segmentation both in the painting of annotation and by training a segmentation model on a small amount of annotation. The support for layers allows multiple datasets to be viewed and annotated together which, for example, enables the use of correlative data (e.g. crowd-sourced annotations or secondary imaging techniques) to guide segmentation. The ability to work with larger data on high-performance servers with GPUs has been added through a client-server architecture and the Pytorch-based image processing and segmentation server is flexible and extensible, and allows the implementation of deep learning-based segmentation modules. The client side has been built around Napari allowing integration of SuRVoS into an ecosystem for open-source image analysis while the server side has been built with cloud computing and extensibility through plugins in mind. Together these improvements to SuRVoS provide a platform for accelerating the annotation and segmentation of volumetric and correlative imaging data across modalities and scales.

Reply to Ngoh et al. Comment on "Surendran et al. The Missed Opportunity of Patient-Centered Medical Homes to Thrive in an Asian Context. Int. J. Environ. Res. Public Health 2021, 18, 1817".

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Homologous Recombination Deficiency: Concepts, Definitions, and Assays.

BACKGROUND: Homologous recombination deficiency (HRD) is a phenotype that is characterized by the inability of a cell to effectively repair DNA double-strand breaks using the homologous recombination repair (HRR) pathway. Loss-of-function genes involved in this pathway can sensitize tumors to poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitors and platinum-based chemotherapy, which target the destruction of cancer cells by working in concert with HRD through synthetic lethality. However, to identify patients with these tumors, it is vital to understand how to best measure homologous repair (HR) status and to characterize the level of alignment in these measurements across different diagnostic platforms. A key current challenge is that there is no standardized method to define, measure, and report HR status using diagnostics in the clinical setting. METHODS: Friends of Cancer Research convened a consortium of project partners from key healthcare sectors to address concerns about the lack of consistency in the way HRD is defined and methods for measuring HR status. RESULTS: This publication provides findings from the group's discussions that identified opportunities to align the definition of HRD and the parameters that contribute to the determination of HR status. The consortium proposed recommendations and best practices to benefit the broader cancer community. CONCLUSION: Overall, this publication provides additional perspectives for scientist, physician, laboratory, and patient communities to contextualize the definition of HRD and various platforms that are used to measure HRD in tumors.

Bcl-xL Enforces a Slow-Cycling State Necessary for Survival in the Nutrient-Deprived Microenvironment of Pancreatic Cancer.

Solid tumors possess heterogeneous metabolic microenvironments where oxygen and nutrient availability are plentiful (fertile regions) or scarce (arid regions). While cancer cells residing in fertile regions proliferate rapidly, most cancer cells in vivo reside in arid regions and exhibit a slow-cycling state that renders them chemoresistant. Here, we developed an in vitro system enabling systematic comparison between these populations via transcriptome analysis, metabolomic profiling, and whole-genome CRISPR screening. Metabolic deprivation led to pronounced transcriptional and metabolic reprogramming, resulting in decreased anabolic activities and distinct vulnerabilities. Reductions in anabolic, energy-consuming activities, particularly cell proliferation, were not simply byproducts of the metabolic challenge, but rather essential adaptations. Mechanistically, Bcl-xL played a central role in the adaptation to nutrient and oxygen deprivation. In this setting, Bcl-xL protected quiescent cells from the lethal effects of cell-cycle entry in the absence of adequate nutrients. Moreover, inhibition of Bcl-xL combined with traditional chemotherapy had a synergistic antitumor effect that targeted cycling cells. Bcl-xL expression was strongly associated with poor patient survival despite being confined to the slow-cycling fraction of human pancreatic cancer cells. These findings provide a rationale for combining traditional cancer therapies that target rapidly cycling cells with those that target quiescent, chemoresistant cells associated with nutrient and oxygen deprivation. SIGNIFICANCE: The majority of pancreatic cancer cells inhabit nutrient- and oxygen-poor tumor regions and require Bcl-xL for their survival, providing a compelling antitumor metabolic strategy.

Scoping review on medical and social domains of ageing research in Singapore (2008-2018).

INTRODUCTION: To examine how ageing research in medical and social domains in Singapore has transformed over time, this scoping review examined the number, types and characteristics of journal publications on ageing in Singapore from 2008 to 2018. METHODS: Using relevant search terms, articles were extracted from multiple databases and then screened and reviewed for eligibility and inclusion by independent reviewers. Data such as title of the study, authors, year of publication, name of journal, type of journal, study design and the kind of data used were charted from the included articles for evidence synthesis. RESULTS: Since 2008, there has been a steady increase in the number of publications on ageing in medical and social domains in Singapore. In the medical domain, publications in Ophthalmology formed the largest group of the existing medical literature on ageing in Singapore (22%), followed by Physical Functioning, which involved physiological measurements of physical well-being (17%) and Geriatrics (16%). Non-medical publications comprised 38% of all the included publications, with publications on the social aspects of ageing forming the largest group in this cluster (43%), followed by publications on Prevention (19%) and Healthcare Services (18%). Most studies were observational in study design (82%), with only 3% of interventional studies. CONCLUSION: While ageing research had expanded in Singapore in the last decade, it was predominantly discipline-specific and observational in design. As ageing issues are complex, with biology intersecting with psychology and sociology, we call for greater interdisciplinary collaboration, interventional studies and more research in understudied and emerging areas.

Cholesterol Auxotrophy as a Targetable Vulnerability in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is characterized by large intracellular lipid droplets containing free and esterified cholesterol; however, the functional significance of cholesterol accumulation in ccRCC cells is unknown. We demonstrate that, surprisingly, genes encoding cholesterol biosynthetic enzymes are repressed in ccRCC, suggesting a dependency on exogenous cholesterol. Mendelian randomization analyses based on 31,000 individuals indicate a causal link between elevated circulating high-density lipoprotein (HDL) cholesterol and ccRCC risk. Depriving ccRCC cells of either cholesterol or HDL compromises proliferation and survival in vitro and tumor growth in vivo; in contrast, elevated dietary cholesterol promotes tumor growth. Scavenger Receptor B1 (SCARB1) is uniquely required for cholesterol import, and inhibiting SCARB1 is sufficient to cause ccRCC cell-cycle arrest, apoptosis, elevated intracellular reactive oxygen species levels, and decreased PI3K/AKT signaling. Collectively, we reveal a cholesterol dependency in ccRCC and implicate SCARB1 as a novel therapeutic target for treating kidney cancer. SIGNIFICANCE: We demonstrate that ccRCC cells are auxotrophic for exogenous cholesterol to maintain PI3K/AKT signaling pathway and ROS homeostasis. Blocking cholesterol import through the HDL transporter SCARB1 compromises ccRCC cell survival and tumor growth, suggesting a novel pharmacologic target for this disease. This article is highlighted in the In This Issue feature, p. 2945.

Perceived facilitators and barriers to chronic disease management in primary care networks of Singapore: a qualitative study.

OBJECTIVE: The increasing chronic disease burden has placed tremendous strain on tertiary healthcare resources in most countries, necessitating a shift in chronic disease management from tertiary to primary care providers. The Primary Care Network (PCN) policy was promulgated as a model of care to organise private general practitioners (GPs) into groups to provide GPs with resources to anchor patients with chronic conditions with them in the community. As PCN is still in its embryonic stages, there is a void in research regarding its ability to empower GPs to manage patients with chronic conditions effectively. This qualitative study aims to explore the facilitators and barriers for the management of patients with chronic conditions by GPs enrolled in PCN. DESIGN: We conducted 30 semistructured interviews with GPs enrolled in a PCN followed by a thematic analysis of audio transcripts until data saturation was achieved. SETTING: Singapore. RESULTS: Our results suggest that PCNs facilitated GPs to more effectively manage patients through (1) provision of ancillary services such as diabetic foot screening, diabetic retinal photography and nurse counselling to permit a 'one-stop-shop', (2) systematic monitoring of process and clinical outcome indicators through a chronic disease registry (CDR) to promote accountability for patients' health outcomes and (3) funding streams for PCNs to hire additional manpower to oversee operations and to reimburse GPs for extended consultations. Barriers include high administrative load in maintaining the CDR due to the lack of a smart electronic clinic management system and financial gradient faced by patients seeking services from private GPs which incur higher out-of-pocket expenses than public primary healthcare institutions. CONCLUSION: PCNs demonstrate great promise in empowering enrolled GPs to manage patients with chronic conditions. However, barriers will need to be addressed to ensure the viability of PCNs in managing more patients in the face of an ageing population.

Validation of a Targeted Next-Generation Sequencing Panel for Tumor Mutation Burden Analysis: Results from the Onconetwork Immuno-Oncology Consortium.

Tumor mutation burden (TMB) is evaluated as a biomarker of response to immunotherapy. We present the efforts of the Onconetwork Immuno-Oncology Consortium to validate a commercial targeted sequencing test for TMB calculation. A three-phase study was designed to validate the Oncomine Tumor Mutational Load (OTML) assay at nine European laboratories. Phase 1 evaluated reproducibility and accuracy on seven control samples. In phase 2, six formalin-fixed, paraffin-embedded samples tested with FoundationOne were reanalyzed with the OTML panel to evaluate concordance and reproducibility. Phase 3 involved analysis of 90 colorectal cancer samples with known microsatellite instability (MSI) status to evaluate TMB and MSI association. High reproducibility of TMB was demonstrated among the sites in the first and second phases. Strong correlation was also detected between mean and expected TMB in phase 1 (r2 = 0.998) and phase 2 (r2 = 0.96). Detection of actionable mutations was also confirmed. In colorectal cancer samples, the expected pattern of MSI-high/high-TMB and microsatellite stability/low-TMB was present, and gene signatures produced by the panel suggested the presence of a POLE mutation in two samples. The OTML panel demonstrated robustness and reproducibility for TMB evaluation. Results also suggest the possibility of using the panel for mutational signatures and variant detection. Collaborative efforts between academia and companies are crucial to accelerate the translation of new biomarkers into clinical research.

Systematic review on the instruments used for measuring the association of the level of multimorbidity and clinically important outcomes.

OBJECTIVES: There are multiple instruments for measuring multimorbidity. The main objective of this systematic review was to provide a list of instruments that are suitable for use in studies aiming to measure the association of a specific outcome with different levels of multimorbidity as the main independent variable in community-dwelling individuals. The secondary objective was to provide details of the requirements, strengths and limitations of these instruments, and the chosen outcomes. METHODS: We conducted the review according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines (PROSPERO registration number: CRD42018105297). We searched MEDLINE, Embase and CINAHL electronic databases published in English and manually searched the Journal of Comorbidity between 1 January 2010 and 23 October 2020 inclusive. Studies also had to select adult patients from primary care or general population and had at least one specified outcome variable. Two authors screened the titles, abstracts and full texts independently. Disagreements were resolved with a third author. The modified Newcastle-Ottawa Scale was used for quality assessment. RESULTS: Ninety-six studies were identified, with 69 of them rated to have a low risk of bias. In total, 33 unique instruments were described. Disease Count and weighted indices like Charlson Comorbidity Index were commonly used. Other approaches included pharmaceutical-based instruments. Disease Count was the common instrument used for measuring all three essential core outcomes of multimorbidity research: mortality, mental health and quality of life. There was a rise in the development of novel weighted indices by using prognostic models. The data obtained for measuring multimorbidity were from sources including medical records, patient self-reports and large administrative databases. CONCLUSIONS: We listed the details of 33 instruments for measuring the level of multimorbidity as a resource for investigators interested in the measurement of multimorbidity for its association with or prediction of a specific outcome.

Vitamin B6 deficiency disrupts serotonin signaling in pancreatic islets and induces gestational diabetes in mice.

In pancreatic islets, catabolism of tryptophan into serotonin and serotonin receptor 2B (HTR2B) activation is crucial for ß-cell proliferation and maternal glucose regulation during pregnancy. Factors that reduce serotonin synthesis and perturb HTR2B signaling are associated with decreased ß-cell number, impaired insulin secretion, and gestational glucose intolerance in mice. Albeit the tryptophan-serotonin pathway is dependent on vitamin B6 bioavailability, how vitamin B6 deficiency impacts ß-cell proliferation during pregnancy has not been investigated. In this study, we created a vitamin B6 deficient mouse model and investigated how gestational deficiency influences maternal glucose tolerance. Our studies show that gestational vitamin B6 deficiency decreases serotonin levels in maternal pancreatic islets and reduces ß-cell proliferation in an HTR2B-dependent manner. These changes were associated with glucose intolerance and insulin resistance, however insulin secretion remained intact. Our findings suggest that vitamin B6 deficiency-induced gestational glucose intolerance involves additional mechanisms that are complex and insulin independent.

The Missed Opportunity of Patient-Centered Medical Homes to Thrive in an Asian Context.

In recent years, there is growing interest internationally to implement patient-centered medical homes (PCMHs), and Singapore is no exception. However, studies understanding the influence of contextual policy factors on the implementation of PCMHs are limited. We conducted 10 semi-structured in-depth interviews with general practitioners working in seven out of the nine PCMHs. Audio recordings were transcribed and analyzed by two study team members in NVivo 12 Software using grounded theory techniques. Power dynamics between the stakeholders and lack of shared decision-making among them in selecting the locale of the PCMH and formulating the practice fee and pharmacy structure were the key factors which negatively affected the implementation of PCMHs on a larger scale. Over time, lack of funding to hire dedicated staff to transfer patients and misalignment of various stakeholders' interest to other right-siting programs also resulted in low number of patients with chronic conditions and revenue. Countries seeking to implement a successful PCMH may benefit from building trust and relationship between stakeholders, engaging in shared decision-making, ongoing cost-efficiency efforts, and formulating a clear delineation of responsibilities between stakeholders. For a healthcare delivery model to succeed in the primary care landscape, policies should be developed keeping mind the realities of primary care practice.

Treating peritoneal dialysis catheter exit-site granulomas with chlorhexidine swabstick: A pilot study.

BACKGROUND: Silver nitrate cauterisation is the conventional treatment for peritoneal dialysis catheter exit-site granulomas. However, it requires to be performed by nurses, patients often experience pain and chemical burns. Therefore, the appropriateness and applicability of using 2% aqueous chlorhexidine swabstick as an alternative was explored in two nephrology centres in Hong Kong. OBJECTIVE: To examine possibility of conducting full trial using chlorhexidine swabstick compared with silver nitrate. DESIGN: A pilot study. PARTICIPANTS: Fort-four patients with exit-site granulomas were equally, randomly allocated to receive chlorhexidine swabstick or silver nitrate. MEASUREMENTS: Both groups were followed for 6 weeks to evaluate the time of granuloma subsidence and adverse effects. Pain and treatment satisfaction were assessed using numerical rating scale and self-developed questionnaire, respectively. RESULTS: Healing rates were 94.4% (17 of 18) using chlorhexidine swabstick,100% (21 of 21) using silver nitrate (p = 0.46). The mean time of granuloma subsidence was significantly longer when using chlorhexidine swabstick (32.8 days) than silver nitrate (12.3 days, p=0.02). The chlorhexidine swabstick group reported significantly fewer adverse effects (11.1%, 2 of 18, p = 0.01) compared with the silver nitrate group (52.4%, 11 of 21). The chlorhexidine swabstick group had lower mean pain score (0.5 of 11) than the silver nitrate group (2.4 of 11, p < 0.01). The satisfaction scores between the two groups had no substantial difference. CONCLUSION: Chlorhexidine swabstick took long time to remove granulomas but had similar success rate, less pain, fewer adverse effects than silver nitrate. Additional research is warranted to examine the applicability of chlorhexidine swabstick.

Comparison of freshwater monitoring approaches: strengths, opportunities, and recommendations.

This review identifies strengths and weaknesses of water monitoring programs selected by Canadian water managers. We used 22 criteria, guided by outcomes of an exploratory study and supported by 21 semi-structured key informant interviews. The highest-scoring programs include the Slave Watershed Environmental Effects Program (Canada), the Government of Canada's Environmental Effects Monitoring Program, and Healthy Land and Water (Australia). We describe five recommendations for improving future freshwater monitoring frameworks: (1) recognize different knowledge approaches (especially Indigenous), (2) use multiple reporting formats, (3) clarify monitoring and management roles, (4) apply a whole-watershed approach, and (5) link monitoring to management and decision-making.

Development of protocols for the first serial block-face scanning electron microscopy (SBF SEM) studies of bone tissue.

There is an unmet need for a high-resolution three-dimensional (3D) technique to simultaneously image osteocytes and the matrix in which these cells reside. In serial block-face scanning electron microscopy (SBF SEM), an ultramicrotome mounted within the vacuum chamber of a microscope repeatedly sections a resin-embedded block of tissue. Backscattered electron scans of the block face provide a stack of high-resolution two-dimensional images, which can be used to visualise and quantify cells and organelles in 3D. High-resolution 3D images of biological tissues from SBF SEM have been exploited considerably to date in the neuroscience field. However, non-brain samples, in particular hard biological tissues, have appeared more challenging to image by SBF SEM due to the difficulties of sectioning and rendering the samples conductive. We have developed and propose protocols for bone tissue preparation using SBF SEM, for imaging simultaneously soft and hard bone tissue components in 3D. We review the state of the art in high-resolution imaging of osteocytes, provide a historical perspective of SBF SEM, and we present first SBF SEM proof-of-concept studies for murine and human tissue. The application of SBF SEM to hard tissues will facilitate qualitative and quantitative 3D studies of tissue microstructure and ultrastructure in bone development, ageing and pathologies such as osteoporosis and osteoarthritis.