Campylobacter jejuni virulence factors: update on emerging issues and trends.

Campylobacter jejuni is a very common cause of gastroenteritis, and is frequently transmitted to humans through contaminated food products or water. Importantly, C. jejuni infections have a range of short- and long-term sequelae such as irritable bowel syndrome and Guillain Barre syndrome. C. jejuni triggers disease by employing a range of molecular strategies which enable it to colonise the gut, invade the epithelium, persist intracellularly and avoid detection by the host immune response. The objective of this review is to explore and summarise recent advances in the understanding of the C. jejuni molecular factors involved in colonisation, invasion of cells, collective quorum sensing-mediated behaviours and persistence. Understanding the mechanisms that underpin the pathogenicity of C. jejuni will enable future development of effective preventative approaches and vaccines against this pathogen.

SATB1 ensures appropriate transcriptional programs within naïve CD8+ T cells.

Special AT-binding protein 1 (SATB1) is a chromatin-binding protein that has been shown to be a key regulator of T-cell development and CD4+ T-cell fate decisions and function. The underlying function for SATB1 in peripheral CD8+ T-cell differentiation processes is largely unknown. To address this, we examined SATB1-binding patterns in naïve and effector CD8+ T cells demonstrating that SATB1 binds to noncoding regulatory elements linked to T-cell lineage-specific gene programs, particularly in naïve CD8+ T cells. We then assessed SATB1 function using N-ethyl-N-nitrosourea-mutant mice that exhibit a point mutation in the SATB1 DNA-binding domain (termed Satb1m1Anu/m1Anu ). Satb1m1Anu/m1Anu mice exhibit diminished SATB1-binding, naïve, Satb1m1Anu/m1Anu CD8+ T cells exhibiting transcriptional and phenotypic characteristics reminiscent of effector T cells. Upon activation, the transcriptional signatures of Satb1m1Anu/m1Anu and wild-type effector CD8+ T cells converged. While there were no overt differences, primary respiratory infection of Satb1m1Anu/m1Anu mice with influenza A virus (IAV) resulted in a decreased proportion and number of IAV-specific CD8+ effector T cells recruited to the infected lung when compared with wild-type mice. Together, these data suggest that SATB1 has a major role in an appropriate transcriptional state within naïve CD8+ T cells and ensures appropriate CD8+ T-cell effector gene expression upon activation.

A technique for preparing undecalcified osteochondral fresh frozen sections for elemental mapping and understanding disease etiology.

The anatomy of the osteochondral junction is complex because several tissue components exist as a unit, including uncalcified cartilage (with superficial, middle, and deep layers), calcified cartilage, and subchondral bone. Furthermore, it is difficult to study because this region is made up of a variety of cell types and extracellular matrix compositions. Using X-ray fluorescence microscopy, we present a protocol for simultaneous elemental detection on fresh frozen samples. We transferred the osteochondral sample using a tape-assisted system and successfully tested it in synchrotron X-ray fluorescence. This protocol elucidates the distinct distribution of elements at the human knee's osteochondral junction, making it a useful tool for analyzing the co-distribution of various elements in both healthy and diseased states.

Differential Angiogenic Potential of 3-Dimension Spheroid of HNSCC Cells in Mouse Xenograft.

The experimental animal model is still essential in the development of new anticancer drugs. We characterized mouse tumors derived from two-dimensional (2D) monolayer cells or three-dimensional (3D) spheroids to establish an in vivo model with highly standardized conditions. Primary cancer-associated fibroblasts (CAFs) were cultured from head and neck squamous cell carcinoma (HNSCC) tumor tissues and co-injected with monolayer cancer cells or spheroids into the oral mucosa of mice. Mice tumor blood vessels were stained, followed by tissue clearing and 3D Lightsheet fluorescent imaging. We compared the effect of exosomes secreted from 2D or 3D culture conditions on the angiogenesis-related genes in HNSCC cells. Our results showed that both the cells and spheroids co-injected with primary CAFs formed tumors. Interestingly, vasculature was abundantly distributed inside the spheroid-derived but not the monolayer-derived mice tumors. In addition, cisplatin injection more significantly decreased spheroid-derived but not monolayer-derived tumor size in mice. Additionally, exosomes isolated from co-culture media of FaDu spheroid and CAF upregulated angiogenesis-related genes in HNSCC cells as compared to exosomes from FaDu cell and CAF co-culture media under in vitro conditions. The mouse tumor xenograft model derived from 3D spheroids of HNSCC cells with primary CAFs is expected to produce reliable chemotherapy drug screening results given the robust angiogenesis and lack of necrosis inside tumor tissues.

Circulating vaccine-derived poliovirus: a menace to the end game of polio eradication.

The World Health Organisation Western Pacific Region countries were declared free of polio in 2000 until a polio outbreak involving 305 cases occurred in Indonesia in 2006. It was not until 2014 that the World Health Organisation South East Asia region was officially declared polio-free again. However, in February 2019, the Global Polio Eradication Initiative announced a new circulating vaccine-derived poliovirus outbreak in the Papua province of Indonesia. To make matter worse, the outbreak responses were tardy and led to transmission among migrating communities to other cities. The pressing regional issues of polio outbreak caused by circulating vaccine-derived poliovirus and use of oral polio vaccine have not been well presented. Our letter highlighted the suboptimal outbreak responses as well as the necessity of cross-border vaccination to curb continued poliovirus transmission.

Seizures Are Associated with Blood-Brain Barrier Disruption in a Piglet Model of Neonatal Hypoxic-Ischaemic Encephalopathy.

Seizures in the neonatal period are most often symptomatic of central nervous system (CNS) dysfunction and the most common cause is hypoxic-ischaemic encephalopathy (HIE). Seizures are associated with poor long-term outcomes and increased neuropathology. Blood-brain barrier (BBB) disruption and inflammation may contribute to seizures and increased neuropathology but are incompletely understood in neonatal HIE. The aim of this study was to investigate the impact of seizures on BBB integrity in a preclinical model of neonatal hypoxic-ischaemic (HI) injury. Piglets (age: <24 h) were subjected to a 30-min HI insult followed by recovery to 72 h post-insult. Amplitude-integrated electroencephalography (aEEG) was performed and seizure burden and background aEEG pattern were analysed. BBB disruption was evaluated in the parietal cortex and hippocampus by means of immunohistochemistry and Western blot. mRNA and protein expression of tight-junction proteins (zonula-occludens 1 [ZO1], occludin [OCLN], and claudin-5 [CLDN5]) was assessed using quantitative polymerase chain reaction (qPCR) and Western blot. In addition, mRNA from genes associated with BBB disruption vascular endothelial growth factor (VEGF) and matrix metalloproteinase 2 (MMP2) as well as inflammatory cytokines and chemokines was assessed with qPCR. Piglets that developed seizures following HI (HI-Sz) had significantly greater injury, as demonstrated by poorer aEEG background pattern scores, lower neurobehavioural scores, and greater histopathology. HI-Sz animals had severe IgG extravasation into brain tissue and uptake into neurons as well as significantly greater levels of IgG in both brain regions as assessed by Western blot. IgG protein in both brain regions was significantly associated with seizure burden, aEEG pattern scores, and neurobehavioural scores. There was no difference in mRNA expression of the tight junctions, however a significant loss of ZO1 and OCLN protein was observed in the parietal cortex. The inflammatory genes TGFß, IL1ß, IL8, IL6, and TNFα were significantly upregulated in HI-Sz animals. MMP2 was significantly increased in animals with seizures compared with animals without seizures. Increasing our understanding of neuropathology associated with seizure is vital because of the association between seizure and poor outcomes. Investigating the BBB is a major untapped area of research and a potential avenue for novel treatments.

Neuropathology in intrauterine growth restricted newborn piglets is associated with glial activation and proinflammatory status in the brain.

BACKGROUND: The fetal brain is particularly vulnerable to intrauterine growth restriction (IUGR) conditions evidenced by neuronal and white matter abnormalities and altered neurodevelopment in the IUGR infant. To further our understanding of neurodevelopment in the newborn IUGR brain, clinically relevant models of IUGR are required. This information is critical for the design and implementation of successful therapeutic interventions to reduce aberrant brain development in the IUGR newborn. We utilise the piglet as a model of IUGR as growth restriction occurs spontaneously in the pig as a result of placental insufficiency, making it a highly relevant model of human IUGR. The purpose of this study was to characterise neuropathology and neuroinflammation in the neonatal IUGR piglet brain. METHODS: Newborn IUGR (< 5th centile) and normally grown (NG) piglets were euthanased on postnatal day 1 (P1; < 18 h) or P4. Immunohistochemistry was utilised to examine neuronal, white matter and inflammatory responses, and PCR for cytokine analysis in parietal cortex of IUGR and NG piglets. RESULTS: The IUGR piglet brain displayed less NeuN-positive cells and reduced myelination at both P1 and P4 in the parietal cortex, indicating neuronal and white matter disruption. A concurrent decrease in Ki67-positive proliferative cells and increase in cell death (caspase-3) in the IUGR piglet brain was also apparent on P4. We observed significant increases in the number of both Iba-1-positive microglia and GFAP-positive astrocytes in the white matter in IUGR piglet brain on both P1 and P4 compared with NG piglets. These increases were associated with a change in activation state, as noted by altered glial morphology. This inflammatory state was further evident with increased expression levels of proinflammatory cytokines (interleukin-1ß, tumour necrosis factor-α) and decreased levels of anti-inflammatory cytokines (interleukin-4 and -10) observed in the IUGR piglet brains. CONCLUSIONS: These findings suggest that the piglet model of IUGR displays the characteristic neuropathological outcomes of neuronal and white matter impairment similar to those reported in the IUGR human brain. The activated glial morphology and elevated proinflammatory cytokines is indicative of an inflammatory response that may be associated with neuronal damage and white matter disruption. These findings support the use of the piglet as a pre-clinical model for studying mechanisms of altered neurodevelopment in the IUGR newborn.

Defects in synaptic transmission at the neuromuscular junction precede motor deficits in a TDP-43Q331K transgenic mouse model of amyotrophic lateral sclerosis.

Transactive response DNA-binding protein-43 (TDP-43) is involved in gene regulation via the control of RNA transcription, splicing, and transport. TDP-43 is a major protein component of ubiquinated inclusions that are found in amyotrophic lateral sclerosis (ALS); however, the function of TDP-43 at the neuromuscular junction (NMJ) and its role in ALS pathogenesis is largely unknown. Here, we show that TDP-43Q331K mutation in mice resulted in impaired neurotransmission by age 3 mo, preceding deficits in motor function and motor neuron loss, which were observed from age 10 mo. These defects were in the effective fusion and release of synaptic vesicles within the motor nerve terminal and manifested in decreased quantal content and reduced probability of quantal release. We observed morphologic alterations that were associated with the TDP-43Q331K mutation, such as aberrant innervation patterns and the distribution of synaptic vesicle-related proteins, which is indicative of a failing NMJ undergoing synaptic remodeling. These findings support a growing acceptance that dysregulation of the NMJ function is a key early event in the pathology of ALS.-Chand, K. K., Lee, K. M., Lee, J. D., Qiu, H., Willis, E. F., Lavidis, N. A., Hilliard, M. A., Noakes, P. G. Defects in synaptic transmission at the neuromuscular junction precede motor deficits in a TDP-43Q331K transgenic mouse model of amyotrophic lateral sclerosis.

Role transition: A descriptive exploratory study of assistant nurse clinicians in Singapore.

AIM: To explore the role-transition experiences of assistant nurse clinicians after their first year of appointment. BACKGROUND: The National Nursing Taskforce was set up in Singapore to examine the professional development and recognition of nurses. It created the assistant nurse clinician role as an avenue for the nurses' career development. The role was intended to assist nurse managers to guide the nursing team in the assessment, planning, and delivery of patient care. METHODS: A qualitative descriptive study design was adopted. A purposive sample of 22 registered nurses from six acute care institutions and two polyclinics in Singapore participated in the face-to-face interviews. An inductive content analysis approach was used to analyse the data. RESULTS: Four themes emerged: (a) promotion to assistant nurse clinician is a form of recognition and vindication; (b) there was uncertainty about the expected role of the assistant nurse clinician; (c) experience eases transition; and (d) there was a need for peer support, mentorship, and training. CONCLUSIONS: The job description of the assistant nurse clinician needs to be better defined to provide greater clarity about their clinical and administrative duties and what is expected of their performance. IMPLICATIONS FOR NURSING MANAGEMENT: It is essential for nurse managers to provide successful role-transition strategies to help the newly appointed assistant nurse clinicians to become efficient and effective leaders.

Loss of laminin-α4 results in pre- and postsynaptic modifications at the neuromuscular junction.

Synaptic basal lamina such as laminin-421 (α4ß2γ1) mediate differentiation of the neuromuscular junction (NMJ). Laminins interact with their pre- or postsynaptic receptors to provide stability and alignment of the pre- to postsynaptic specializations. Knockout of the laminin-α4 gene (lama4) does not alter gross NMJ morphogenesis. However, mice deficient in laminin-α4 (lama4-/- mice) display disruptions in the alignment of the active zones and postsynaptic folds at the NMJ, although the physiological consequences of this loss have not been examined. The present study investigated the differences in neurotransmission during the early development and maturation of the NMJ in lama4-/- and wild-type mice. Lama4-/- NMJs demonstrated a decrease in miniature end-plate potential (EPP) frequency and increased amplitude of miniature EPPs and evoked EPPs. Binomial parameters analysis of neurotransmitter release revealed a decrease in quantal release, the result of a decrease in the number of active release sites, but not in the probability of transmitter release. Lama4-/- NMJs displayed higher levels of synaptic depression under high-frequency stimulation and altered facilitation, suggesting compromised delivery of synaptic vesicles. This idea is supported by our molecular investigations of lama4-/- NMJs, where we see altered distribution of Bassoon, a molecular component of active zones, presumably resulting from perturbed neurotransmission.-Chand, K. K., Lee, K. M., Lavidis, N. A., Noakes, P. G. Loss of laminin-α4 results in pre- and postsynaptic modifications at the neuromuscular junction.

Pricing appraisal of anti-cancer drugs in the South East Asian, Western Pacific and East Mediterranean Region.

BACKGROUND: Globally, cancer is one of the leading causes of mortality. High treatment cost, partly owing to higher prices of anti-cancer drugs, presents a significant burden on patients and healthcare systems. The aim of the present study was to survey and compare retail prices of anti-cancer drugs between high, middle and low income countries in the South-East Asia, Western Pacific and Eastern Mediterranean regions. METHODS: Cross-sectional survey design was used for the present study. Pricing data from ten counties including one from South-East Asia, two from Western Pacific and seven from Eastern Mediterranean regions were used in this study. Purchasing power parity (PPP)-adjusted mean unit prices for 26 anti-cancer drug presentations (similar pharmaceutical form, strength, and pack size) were used to compare prices of anti-cancer drugs across three regions. A structured form was used to extract relevant data. Data were entered and analysed using Microsoft Excel®. RESULTS: Overall, Taiwan had the lowest mean unit prices while Oman had the highest prices. Six (23.1%) and nine (34.6%) drug presentations had a mean unit price below US$100 and between US$100 and US$500 respectively. Eight drug presentations (30.7%) had a mean unit price of more than US$1000 including cabazitaxel with a mean unit price of $17,304.9/vial. There was a direct relationship between income category of the countries and their mean unit price; low-income countries had lower mean unit prices. The average PPP-adjusted unit prices for countries based on their income level were as follows: low middle-income countries (LMICs): US$814.07; high middle income countries (HMICs): US$1150.63; and high income countries (HICs): US$1148.19. CONCLUSIONS: There is a great variation in pricing of anticancer drugs in selected countires and within their respective regions. These findings will allow policy makers to compare prices of anti-cancer agents with neighbouring countries and develop policies to ensure accessibility and affordability of anti-cancer drugs.

Loss of ß2-laminin alters calcium sensitivity and voltage-gated calcium channel maturation of neurotransmission at the neuromuscular junction.

KEY POINTS: Neuromuscular junctions from ß2-laminin-deficient mice exhibit lower levels of calcium sensitivity. Loss of ß2-laminin leads to a failure in switching from N- to P/Q-type voltage-gated calcium channel (VGCC)-mediated transmitter release that normally occurs with neuromuscular junction maturation. The motor nerve terminals from ß2-laminin-deficient mice fail to up-regulate the expression of P/Q-type VGCCs clusters and down-regulate N-type VGCCs clusters, as they mature. There is decreased co-localisation of presynaptic specialisations in ß2-laminin-deficient neuromuscular junctions as a consequence of lesser P/Q-type VGCC expression. These findings support the idea that ß2-laminin is critical in the organisation and maintenance of active zones at the neuromuscular junction via its interaction with P/Q-type VGCCs, which aid in stabilisation of the synapse. ß2-laminin is a key mediator in the differentiation and formation of the skeletal neuromuscular junction. Loss of ß2-laminin results in significant structural and functional aberrations such as decreased number of active zones and reduced spontaneous release of transmitter. In vitro ß2-laminin has been shown to bind directly to the pore forming subunit of P/Q-type voltage-gated calcium channels (VGCCs). Neurotransmission is initially mediated by N-type VGCCs, but by postnatal day 18 switches to P/Q-type VGCC dominance. The present study investigated the changes in neurotransmission during the switch from N- to P/Q-type VGCC-mediated transmitter release at ß2-laminin-deficient junctions. Analysis of the relationship between quantal content and extracellular calcium concentrations demonstrated a decrease in the calcium sensitivity, but no change in calcium dependence at ß2-laminin-deficient junctions. Electrophysiological studies on VGCC sub-types involved in transmitter release indicate N-type VGCCs remain the primary mediator of transmitter release at matured ß2-laminin-deficient junctions. Immunohistochemical analyses displayed irregularly shaped and immature ß2-laminin-deficient neuromuscular junctions when compared to matured wild-type junctions. ß2-laminin-deficient junctions also maintained the presence of N-type VGCC clustering within the presynaptic membrane, which supported the functional findings of the present study. We conclude that ß2-laminin is a key regulator in development of the NMJ, with its loss resulting in reduced transmitter release due to decreased calcium sensitivity stemming from a failure to switch from N- to P/Q-type VGCC-mediated synaptic transmission.

Association of UCP1 -3826A/G and UCP3 -55C/T gene polymorphisms with obesity and its related traits among multi-ethnic Malaysians.

OBJECTIVE: Our study investigated the association of UCP1 -3826A/G and UCP3 -55C/T single nucleotide polymorphisms (SNPs) with obesity and its related traits among multi-ethnic Malaysians. PARTICIPANTS: A total of 447 (225 males; 46 Malays, 339 ethnic Chinese, 62 ethnic Indians; 111 obese) participated. METHODS: Demographic and anthropometric data were collected, and genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: The minor allele frequencies (MAFs) for UCP1 according to Malay/Chinese/Indian ethnicities were .61/.55/.52 and .32/.55/.38, respectively. UCP3 genotype and allele distribution was significantly associated with ethnicity and waist-to-hip ratio (WHR), but among non-obese and Chinese participants only, respectively, after stratified analysis. Chinese participants with T allele had significantly lesser risk to be centrally obese [odds ratio =.69 (CI =.48, 1.00; P=.04)], and also had significantly lower WHR compared to those with C allele. The UCP1 or UCP3 SNPs were not associated with obesity/BMI and total body fat (TBF), but combinatory genotype analysis revealed that those having the AA and CC genotype for the former and latter SNPs had significantly highest BMI and TBF compared to other genotype combinations. CONCLUSIONS: UCP3 -55C/T SNP was associated with central obesity among Malaysian participants of Chinese descent. Combinatory genotype analysis showed that BMI and TBF were significantly different among UCPI -3826A/G and UCP3 -55C/T genotype combinations, suggesting the existence of a gene interaction between UCP1 and UCP3 in influencing obesity and adiposity.

The detection, characterization, and quantification of dominant degradation products of nisin A and Z in selected dairy products by liquid chromatography-high-resolution mass spectrometry technique.

Nisin, a bacteriocin produced through fermentation using bacterium Lactococcus lactis, has several commercial variants such as nisin A and nisin Z. Nisin serves as a natural preservative with antimicrobial properties in various food products, including dairy and beverages, for extending product shelf life. The efficacy and safety of nisin A as a bacteriocin has been well characterized. However, there is limited evidence regarding the efficacy, stability, and safety of nisin Z as a food preservative, as it has not undergone comprehensive regulatory reviews. In this work, we studied the stability of nisin A and Z in a selection of yogurt drinks and found nisin to be unstable, particularly in fruit-flavored yogurt drinks. Both nisin A and Z could experience significant degradation leading to the nisin parent ion peaks dropping below detectable level before the product's expiry date. Compared with nisin A, the formation of oxidized metabolite nisin Z+O appeared to be the predominant reaction for nisin Z. These findings highlight the need for further scientific research to understand the behavior of nisin Z under different application conditions, which is crucial for assessing the efficacy and safety of nisin Z under these conditions. One potential application of this knowledge is to optimize the formulation of yogurt-based drinks to stabilize nisin Z and sustain its biopreservative function throughout the product's shelf life. Additionally, the current study shows that for the testing of the presence of nisin A or nisin Z, it is imperative to cover both the parent and the main degradant(s) of nisin. This is especially true for nisin Z, for which the regulatory approval status may vary in different markets. As such, the confirmative identification of nisin Z and its key metabolites in commercial products would be essential.

Differential effect of cancer-associated fibroblast-derived extracellular vesicles on cisplatin resistance in oral squamous cell carcinoma via miR-876-3p.

Rationale: Platinum-based chemotherapy is commonly used for treating solid tumors, but drug resistance often limits its effectiveness. Cancer-associated fibroblast (CAF)-derived extracellular vesicle (EV), which carry various miRNAs, have been implicated in chemotherapy resistance. However, the molecular mechanism through which CAFs modulate cisplatin resistance in oral squamous cell carcinoma (OSCC) is not well understood. We employed two distinct primary CAF types with differential impacts on cancer progression: CAF-P, representing a more aggressive cancer-promoting category, and CAF-D, characterized by properties that moderately delay cancer progression. Consequently, we sought to investigate whether the two CAF types differentially affect cisplatin sensitivity and the underlying molecular mechanism. Methods: The secretion profile was examined by utilizing an antibody microarray with conditioned medium obtained from the co-culture of OSCC cells and two types of primary CAFs. The effect of CAF-dependent factors on cisplatin resistance was investigated by utilizing conditioned media (CM) and extracellular vesicle (EVs) derived from CAFs. The impacts of candidate genes were confirmed using gain- and loss-of-function analyses in spheroids and organoids, and a mouse xenograft. Lastly, we compared the expression pattern of the candidate genes in tissues from OSCC patients exhibiting different responses to cisplatin. Results: When OSCC cells were cultured with conditioned media (CM) from the two different CAF groups, cisplatin resistance increased only under CAF-P CM. OSCC cells specifically expressed insulin-like growth factor binding protein 3 (IGFBP3) after co-culture with CAF-D. Meanwhile, IGFBP3-knockdown OSCC cells acquired cisplatin resistance in CAF-D CM. IGFBP3 expression was promoted by GATA-binding protein 1 (GATA1), a transcription factor targeted by miR-876-3p, which was enriched only in CAF-P-derived EV. Treatment with CAF-P EV carrying miR-876-3p antagomir decreased cisplatin resistance compared to control miRNA-carrying CAF-P EV. On comparing the staining intensity between cisplatin-sensitive and -insensitive tissues from OSCC patients, there was a positive correlation between IGFBP3 and GATA1 expression and cisplatin sensitivity in OSCC tissues from patients. Conclusion: These results provide insights for overcoming cisplatin resistance, especially concerning EVs within the tumor microenvironment. Furthermore, it is anticipated that the expression levels of GATA1 and miR-876-3p, along with IGFBP3, could aid in the prediction of cisplatin resistance.

Singapore's Total Diet Study (2021-2023): Study Design, Methodology, and Relevance to Ensuring Food Safety.

A total diet study is often used to evaluate a population's baseline dietary exposure to chemical hazards from across the diet. In 2021-2023, Singapore carried out a TDS, and this article presents an overview of the study design and methodological selections in Singapore's TDS, as well as its relevance to ensuring food safety. A food consumption survey was conducted on Singapore citizens and permanent residents, where food consumption patterns of the Singapore population were identified. The selection of chemical hazards and foods for inclusion in Singapore's TDS, as well as principal considerations on sampling, food preparation, and analytical testing are discussed. Commonly consumed foods by the Singapore population in food categories such as grain and grain-based products, meat and meat products, fish and seafood, vegetables, fruits, milk and dairy products were included in this study, and mean concentrations of chemicals tested in each food category were reported, with food categories possessing higher levels identified. Future work will include dietary exposure assessments for the population and analysis of the contributions by food and cooking method.

Chromogenic in situ hybridization technique for detecting porcine circovirus 3 in lung and lymphoid tissues.

Background and Aim: Porcine circovirus 3 (PCV3) was recently reported in Malaysian commercial pig population in 2020 by conventional polymerase chain reaction (PCR), revealing a molecular prevalence of 17.02% in the sampled domestic pig population. This study aims to describe a chromogenic in situ hybridization (ISH) technique using digoxigenin (DIG)-labeled cloned PCV3 open reading frame 1 (ORF1) fragment DNA to detect and localize the PCV3 antigen in formalin-fixed, paraffin-embedded lung, and lymphoid tissue specimens. Materials and Methods: Since PCV3 was mainly detected in lung and lymphoid tissues, we obtained tissue specimens from these organs from the previous Malaysian PCV3 study. Digoxigenin-labeled ISH probes were designed to target a 69 bp region of PCV3 ORF1 spanning from the nucleotide positions (282-350). Results: Light microscopy analysis revealed that chromogenic staining of PCV3 antigens was visualized within the cytoplasm of pneumocytes and lymphocytes, indicating positive ISH results. The results of molecular detection of PCV3 using PCR and ISH showed a high agreement of 90.91%, including for the negative PCV3 status for all samples. Conclusion: This study reports a chromogenic ISH technique using DIG-labeled probes targeting PCV3 ORF1 to detect PCV3 antigens in lung and lymphoid tissues. Despite the limited availability of PCV3 antibodies, ISH remains relevant for investigating PCV3 replication and pathogenesis and can be used complementarily with PCR for evaluating the localization of antigens in infected tissues.

Evaluating healthcare professionals' perceptions of dispensing separation and sale of pharmacy medicine in Brunei Darussalam.

BACKGROUND: Pharmacy medicine (P) is obtained exclusively from a pharmacy under the supervision of a pharmacist. This study aims to understand the perception of healthcare professionals towards the dispensing separation, as well as the dispensing of pharmacy medicine by community pharmacies to enhance patient health outcomes in Brunei Darussalam. METHODS: A cross-sectional study was conducted between 1st March 2023 and 20th April 2023 among healthcare professionals. A newly designed and validated questionnaire was used. Its face and content validity, along with internal consistency, was adequately established. Convenient sampling was employed to recruit participants for the study. Statistical analysis using one-way ANOVA was performed, considering a p-value < 0.05 as statistically significant. RESULTS: The study compiled data from 108 participants, comprising doctors (38.9%) and pharmacy technicians (45.4%). Approximately 28.7% of respondents had 11-20 years of healthcare experience, while 25.9% had less than 5 years. Nearly all respondents (98.1%) agreed on the vital role pharmacists and pharmacy technicians play in prescription checks. A significant number of participants (93.5%) agreed that Brunei's current medicine dispensing system needs improvement. The mean total score for the perception of medicine dispensing in Brunei was 3.79 ± 1.103. A statistically significant difference was found between the perception score and the respondents' profession (p = 0.018), but not with their age, experience, or place of work. Respondents' awareness score showed no statistically significant correlation with their profession, age, experience, or place of work. CONCLUSION: The study underscores the necessity for more patient-centered care in community pharmacies in Brunei Darussalam. The country's healthcare professionals should recognize the potential advantages of expanding pharmacy services. However, to implement these services successfully, regulatory restrictions and infrastructure limitations must be addressed.

Utility and usability evaluation of an information diary tool to measure health information access and exposure among patients with high cardiovascular risk.

Background: Online health misinformation about statins potentially affects health decision-making on statin use and adherence. We developed an information diary platform (IDP) to measure topic-specific health information exposure where participants record what information they encounter. We evaluated the utility and usability of the smartphone diary from the participants' perspective. Methods: We used a mixed-method design to evaluate how participants used the smartphone diary tool and their perspectives on usability. Participants were high cardiovascular-risk patients recruited from a primary care clinic and used the tool for a week. We measured usability with the System Usability Scale (SUS) questionnaire and interviewed participants to explore utility and usability issues. Results: The information diary was available in three languages and tested with 24 participants. The mean SUS score was 69.8 ± 12.9. Five themes related to utility were: IDP functions as a health information diary; supporting discussion of health information with doctors; wanting a feedback function about credible information; increasing awareness of the need to appraise information; and wanting to compare levels of trust with other participants or experts. Four themes related to usability were: ease of learning and use; confusion about selecting the category of information source; capturing offline information by uploading photos; and recording their level of trust. Conclusion: We found that the smartphone diary can be used as a research instrument to record relevant examples of information exposure. It potentially modifies how people seek and appraise topic-specific health information.