The Mycobacterium tuberculosis (Mtb) genome is over 4.4 million base pairs long and contains about 4,400 genes. While many bioinformatics tools exist for studying bacterial genomes, few are attuned ...to peculiarities of Mtb. Repetitive regions and the genomic diversity among clinical isolates warrant special attention when doing computational genomic research on this pathogen. Here, I present my contributions to the advancement of two bioinformatics tools written in Python and to a SQLite database, all of which are focused around studying variants in Mtb. The first tool, PBHoover, is a heterogeneity-aware variant caller that, given enough read depth and quality, can identify genomic subpopulations. Until now it was only able to accept the deprecated cmph5 format as input, limiting its usefulness as new alignment data is generated in the ubiquitous BAM format. I have modified the codebase to allow it to accept BAM files, and to call variants with these files in a reasonable amount of time. The second tool, Hybran, combines ab-initio and reference-based methods to annotate genomic features in the Mtb genome. Hybran annotates 485 novel open reading frames (ORFs) across 110 genomes but not the reference H37Rv. An additional 18 are annotated in each clinical isolate genome as well as H37Rv. I have used RNA-seq data, comparison to a manual annotation, and a homology analysis to attempt to validate thesenovel ORFs, finding strong evidence of protein expression in at least 25 of the 503 totalnovel ORFs. Finally, I built a SQLite database to store genomic variant information andmetadata for 492 clinical Mtb isolates. These tools should prove useful for our lab and otherswho are studying the Mtb genome with computational methods.
Dengue neurological disease is an uncommon yet severe complication of dengue infection. It can manifest as encephalitis, encephalopathy, neuro-ophthalmic complications, or neuromuscular disorders. ...Severe infection can result in viral shedding across multiple body sites. We describe a case of severe neuro-ophthalmic dengue infection in an otherwise healthy returned traveller, presenting with prolonged multiple-body-site viral detections by PCR. The dengue virus (DENV) dynamics and serological response support a direct DENV neuropathogenicity. A retrospective review of the laboratory data at the Victorian Infectious Diseases Reference Laboratory (VIDRL) suggests that blood is the most frequent sample type with DENV detection (92% of all DENV-positive samples). Genotype variation is seen across different sample types. The similarity of CSF and nasopharyngeal DENV subtypes (genotype 1 and 3) suggests a possible correlation between nasopharyngeal replication and neurological complications. The case presented highlights the direct neuropathogenicity of DENV early in the course of infection, and a potential correlation between nasopharyngeal replication and neurological disease.
Abstract
Background
During the Omicron BA.1 surge between 12 January and 18 February 2022, 189 COVID-19-positive pregnant women were managed by the Peninsula Health Positive Pathways program, in the ...Mornington Peninsula, Victoria, Australia. A multidisciplinary specialist team was rapidly assembled to work in conjunction with the Pathways COVID-19 physicians and monitoring team, to optimize patient care.
Objective
The aim is to describe the processes utilized to care for the pregnancy cohort from the time of enrolment to the COVID Monitor until recovery including outpatient monitoring, treatment strategies, hospital review criteria, and clinical outcomes including rate of hospitalization, oxygen requirements, and maternal and foetal outcomes during the study period.
Method
Outpatients were monitored daily by the Pathways program, while COVID-19 physicians and obstetricians conducted early telehealth review of patients after diagnosis. Members of the multidisciplinary team met on a virtual platform twice daily, and institution-specific treatment guidelines and hospital review criteria were established. Enoxaparin prophylaxis was delivered to the homes of selected patients, and inhaled budesonide was utilized for patients who did not require oxygen, who were immunocompromised, and who had significant respiratory symptoms or risk factors for deterioration. Sotrovimab was offered to women as per the existing Australian National COVID-19 Clinical Evidence Taskforce eligibility criteria. A service evaluation was undertaken adopting a retrospective cohort approach.
Results
There was minimal maternal morbidity and no mortality with 24/189 (12.7%) women requiring hospitalization, 18/189 (9.5%) requiring same-day emergency department presentations only, and 4/189 (2.1%) requiring oxygen, with no requirement for non-invasive ventilation or intensive care unit admission. Sixteen patients delivered live newborns during the study period, and there were two pregnancy losses at 7 and 19 weeks gestation, respectively, in patients with prior pregnancy complications.
Conclusion
A multidisciplinary approach involving virtual communication twice daily between treating specialist physicians may be a broadly applicable model to optimize care of pregnant women with COVID-19.
Background:
This quality improvement study, entitled Avatar-Based LEarning for Diabetes Optimal Control (ABLEDOC), explored the feasibility of delivering an educational program to people with ...diabetes in Colombia. The aim was to discover how this approach could be used to improve awareness and understanding of the condition, the effects of treatment, and strategies for effective management of blood-glucose control.
Methods:
Individuals with diabetes were recruited by Colombian endocrinologists to a human-centered study to codesign the educational program, using the Double Diamond model. Participants contributed to two phases. The first phase focused on gathering unmet educational needs and choice of curriculum. Three prototypes were developed as a result. During phase 2, a different group of participants engaged with the program for several weeks, before reporting back.
Results:
Thirty-six participants completed a Web survey during phase 1, and five were also interviewed by telephone. The majority (33 of 36; 91%) were receptive to the prospect of educational interventions and ranked the chosen topic of hypoglycemia highly. In phase 2, the three prototypes were tested by 17 participants, 10 of whom also gave feedback in focus groups. The response was overwhelmingly positive, with 16 of 17 (94%) stating they would use a program like this again. The 3D version was the most highly rated.
Conclusions:
Immersive, avatar-based programs, delivered through smartphone, have the potential to deliver educational information that is trusted, engaging, and useful. Future work includes expansion of the curriculum, evaluation with a larger group, and exploration of the prospective role of artificial intelligence in personalizing this form of educational intervention.
The longevity of an organism is influenced by both genetic and environmental factors. With respect to genetic factors, a significant effort is being made to identify pharmacological agents that ...extend life span by targeting pathways with a defined role in the aging process. On the environmental side, the molecular mechanisms responsible for the positive influence of interventions such as dietary restriction are being explored. The environment experienced by humans in modern societies already contains countless compounds that may influence longevity. Understanding the role played by common compounds that substantially affect the aging process will be critical for predicting and interpreting the outcome of introducing new interventions. Caffeine is the most widely used psychoactive drug worldwide. Prior studies in flies, worms, and mice indicate that caffeine may positively impact age-associated neurodegenerative pathology, such as that observed in Alzheimer's disease.
Here we report that caffeine is capable of extending life span and improving healthspan in Caenorhabditis elegans, a finding that is in agreement with a recently published screen looking for FDA-approved compounds capable of extending worm life span. Life span extension using caffeine displays epistatic interaction with two known longevity interventions: dietary restriction and reduced insulin signaling. Caffeine treatment also delays pathology in a nematode model of polyglutamine disease.
The identification of caffeine as a relevant factor in aging and healthspan in worms, combined with prior work in both humans and rodents linking caffeine consumption to reduced risk of age-associated disease, suggests that caffeine may target conserved longevity pathways. Further, it may be important to consider caffeine consumption when developing clinical interventions, particularly those designed to mimic dietary restriction or modulate insulin/IGF-1-like signaling. The positive impact of caffeine on a worm model of polyglutamine disease suggests that chronic caffeine consumption may generally enhance resistance to proteotoxic stress and may be relevant to assessing risk and developing treatments for human diseases like Alzheimer's and Huntington's disease. Future work addressing the relevant targets of caffeine in models of aging and healthspan will help to clarify the underlying mechanisms and potentially identify new molecular targets for disease intervention.
This study assessed the toxicological and biological responses of aerosols from a novel hybrid tobacco product. Toxicological responses from the hybrid tobacco product were compared to those from a ...commercially available Tobacco Heating Product (c-THP), a prototype THP (p-THP) and a 3R4F reference cigarette, using in vitro test methods which were outlined as part of a framework to substantiate the risk reduction potential of novel tobacco and nicotine products. Exposure matrices used included total particulate matter (TPM), whole aerosol (WA), and aqueous aerosol extracts (AqE) obtained after machine-puffing the test products under the Health Canada Intense smoking regime. Levels of carbonyls and nicotine in these matrices were measured to understand the aerosol dosimetry of the products. The hybrid tobacco product tested negative across the in vitro assays including mutagenicity, genotoxicity, cytotoxicity, tumour promotion, oxidative stress and endothelial dysfunction. All the THPs tested demonstrated significantly reduced responses in these in vitro assays when compared to 3R4F. The findings suggest these products have the potential for reduced health risks. Further pre-clinical and clinical assessments are required to substantiate the risk reduction of these novel products at individual and population levels.
•Aerosol produced from a novel hybrid tobacco product was assessed for biological activity using a suite of in vitro assays.•The in vitro responses were compared with those induced by tobacco-heating products and a reference cigarette.•In vitro dosimetry methods were applied to assess the levels of key toxicants in the aerosol exposure matrices.•The hybrid tobacco product demonstrated significant reductions in biological activity compared to the reference cigarette.•The hybrid tobacco product also showed lower activity than tobacco-heating products in many of these tests.
Abstract Background: Gastrointestinal (GI) cancers cause more cancer deaths than any other body system, with 3.4 million related deaths in 2018. Wnt pathway activation is common in GI cancers, with ...the pathogenic upstream Wnt pathway variant sub-population (RNF43 loss of function/RSPO gain of function) showing a combined prevalence of 4.7% in GI cancer patients; pre-clinically this sub-population shows exquisite sensitivity to RXC004, a small molecule Porcupine inhibitor. A striking co-occurrence of upstream Wnt pathway variants with MAPK pathway driver mutations (KRAS, BRAF, NRAS) was detected in 77% of Microsatellite stable (MSS) GI cancers, suggesting co-inhibiting these pathways could enhance clinical activity in these patients. Methods: In vitro proliferation assays were performed in pre-clinical models of upstream Wnt pathway mutated MSS GI cancer (SNU-1411, HPAF-II and AsPC-1) using Wnt (RXC004) or MAPK (Trametinib) pathway inhibitors at multiple concentrations as single agents or in combination. Control cells with downstream Wnt pathway mutations (WiDr and HCT116) were also tested. Synergistic combination effects were detected by BLISS scores. RXC004 (1.5mg/kg QD) and Trametinib (0.3mg/kg QD) were evaluated in an RSPO-fusion xenograft model (SNU-1411) as single agents or in combination. Efficacy was measured by tumour volume and weight; PD markers were assessed in end of study tumour samples. Relative AXIN2 and DUSP6 gene expression changes were determined upon inhibitor treatment by quantitative Polymerase Chain Reaction (qPCR). Results: RXC004 in combination with Trametinib demonstrated strong anti-proliferative synergy in SNU-1411 cells and moderate synergy/additivity in HPAF-II and AsPC-1 cells; no synergy was observed in control cells. In vivo, RXC004 and Trametinib monotherapy led to significant moderate tumour growth control (p<0.05) at the doses tested, whereas their combination led to significantly enhanced efficacy (p<0.05). Gene expression analysis demonstrated a reciprocal Wnt/MAPK signaling mechanism; RXC004 strongly suppressed Wnt signaling (AXIN2) but increased MAPK signaling (DUSP6), whilst Trametinib strongly suppressed MAPK signaling (DUSP6) but increased Wnt signaling (AXIN2). Combination of RXC004 and Trametinib sustained inhibition of both Wnt and MAPK signalling. Similar effects were observed with inhibitors of other MAPK pathway components. Conclusion: Wnt and MAPK pathways are frequently co-activated in GI cancers, particularly in the upstream Wnt pathway mutated sub-population. Pre-clinically we show that Wnt and MAPK pathways act as potential reciprocal resistance mechanisms following single agent inhibition of either pathway; co-inhibition of these pathways leads to synergistic effects in vitro and enhanced efficacy in vivo. This provides a clear rationale to assess this combination approach in the clinic. Citation Format: Simon A. Woodcock, Dorottya Keppel, Catherine Eagle, James Kelly, Eimear Flanagan, Emma Bishop, Inder Bhamra, Clifford D. Jones, Richard Armer, Jane Robertson, Caroline Phillips. Pre-clinical activity of the Wnt pathway inhibitor RXC004 in combination with MAPK pathway inhibitors in GI cancer models abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 944.
The use of reconstituted human airway (RHuA) epithelial tissues to assess functional endpoints is highly relevant in respiratory toxicology, but standardised methods are lacking. In June 2015, the ...Institute for In Vitro Sciences (IIVS) held a technical workshop to evaluate the potential for standardisation of methods, including ciliary beat frequency (CBF). The applicability of a protocol suggested in the workshop was assessed in a multi-laboratory ring study. This report summarises the findings, and uses the similarities and differences identified between the laboratories to make recommendations for researchers in the absence of a validated method. Two software platforms for the assessment of CBF were used — Sisson-Ammons Video Analysis (SAVA; Ammons Engineering, Clio, MI, USA) and ciliaFA (National Institutes of Health, Bethesda, MD, USA). Both were utilised for multiple read temperatures, one objective strength (10×) and up to four video captures per tissue, to assess their utility. Two commercial RHuA tissue cultures were used: MucilAir™ (Epithelix, Geneva, Switzerland) and EpiAirway™ (MatTek, Ashland, MA, USA). IL-13 and procaterol were used to induce CBF-specific responses as positive controls. Further testing addressed the impact of tissue acclimation duration, the number of capture fields and objective strengths on baseline CBF readings. Both SAVA and ciliaFA reliably collected CBF data. However, ciliaFA failed to generate accurate CBF measurements above ∼10 Hz. The positive controls were effective, but were subject to inter-laboratory variability. CBF endpoints were generally uniform across replicate tissues, objective strengths and laboratories. Longer tissue acclimation increased the percentage active area, but had minimal impact on CBF. Taken together, these findings support the development and validation of a standardised CBF measurement protocol.
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