Controlled release of vancomycin from PEGylated fibrinogen polyethylene glycol diacrylate hydrogel.

Surgical site infection (SSI) is a common issue post-surgery which often prolongs hospitalization and can lead to serious complications such as sternal wound infection following cardiac surgery via median sternotomy. Controlled release of suitable antibiotics could allow maximizing drug efficacy and safety, and therefore achieving a desired therapeutic response. In this study, we have developed a vancomycin laden PEGylated fibrinogen-polyethylene glycol diacrylate (PF-PEGDA) hydrogel system that can release vancomycin at a controlled and predictable rate to be applied in SSI prevention. Two configurations were developed to study effect of the hydrogel on drug release, namely, vancomycin laden hydrogel and vancomycin solution on top of blank hydrogel. The relationship between the rigidity of the hydrogel and drug diffusion was found to comply with a universal power law, i.e., softer hydrogels result in a greater diffusion coefficient hence faster release rate. Besides, vancomycin laden hydrogels exhibited burst release, whereas the vancomycin solution on top of blank hydrogels exhibited lag release. A mathematical model was developed to simulate vancomycin permeation through the hydrogels. The permeation of vancomycin can be predicted accurately by using the mathematical model, which provided a useful tool to customize drug loading, hydrogel thickness and stiffness for personalized medication to manage SSI. To evaluate the potential of hydrogels for bone healing applications in cardiovascular medicine, we performed a proof-of-concept median sternotomy in rabbits and applied the hydrogels. The hydrogel formulations accelerated the onset of osteo-genetic processes in rabbits, demonstrating its potential to be used in human.

Highlight of 2023: big impacts of microRNAs in T cells.

In 2023, several significant discoveries on the function of microRNAs in the immune system were reported. Here we discuss several notable papers that revealed important functions in T cells.

Non-canonical RNA substrates of Drosha lack many of the conserved features found in primary microRNA stem-loops.

The RNase III enzyme Drosha has a central role in microRNA (miRNA) biogenesis, where it is required to release the stem-loop intermediate from primary (pri)-miRNA transcripts. However, it can also cleave stem-loops embedded within messenger (m)RNAs. This destabilizes the mRNA causing target gene repression and appears to occur primarily in stem cells. While pri-miRNA stem-loops have been extensively studied, such non-canonical substrates of Drosha have yet to be characterized in detail. In this study, we employed high-throughput sequencing to capture all polyA-tailed RNAs that are cleaved by Drosha in mouse embryonic stem cells (ESCs) and compared the features of non-canonical versus miRNA stem-loop substrates. mRNA substrates are less efficiently processed than miRNA stem-loops. Sequence and structural analyses revealed that these mRNA substrates are also less stable and more likely to fold into alternative structures than miRNA stem-loops. Moreover, they lack the sequence and structural motifs found in miRNA stem-loops that are required for precise cleavage. Notably, we discovered a non-canonical Drosha substrate that is cleaved in an inverse manner, which is a process that is normally inhibited by features in miRNA stem-loops. Our study thus provides valuable insights into the recognition of non-canonical targets by Drosha.

Mapping the two distinct proliferative bursts early in T-cell development.

T-cell development occurs in the thymus and is tightly regulated to produce a diverse enough repertoire of mature T cells that can recognize any potential pathogen. The development of T cells is dependent on small numbers of uncommitted precursors that continually seed the thymus from the bone marrow. As they progress along the developmental pathway, there is a massive expansion in cell number to generate the necessary diversity in T-cell receptor chain usage. It is recognized that there are two proliferative bursts that occur early in T-cell development, one prior to ß-selection and one after, and these are responsible for the expansion. While the proliferation following ß-selection is well-characterized, the earlier proliferative burst has yet to be precisely defined. In this study, we employ single-cell RNA sequencing coupled to trajectory inference methods to pinpoint when in T-cell development thymocytes are induced into cell cycle. We show that the first proliferative burst is initiated in the double-negative (DN) 2a stage before T lineage commitment occurs, with cell cycling downregulated by the DN3a stage. A second burst is then initiated at the DN3b stage, immediately after ß-selection. We subsequently employ fluorescence-activated cell sorting-based analysis for DNA content to confirm these two proliferative bursts.

Distinct subpopulations of DN1 thymocytes exhibit preferential γδ T lineage potential.

The αß and γδ T cell lineages both differentiate in the thymus from common uncommitted progenitors. The earliest stage of T cell development is known as CD4-CD8- double negative 1 (DN1), which has previously been shown to be a heterogenous mixture of cells. Of these, only the CD117+ fraction has been proposed to be true T cell progenitors that progress to the DN2 and DN3 thymocyte stages, at which point the development of the αß and γδ T cell lineages diverge. However, recently, it has been shown that at least some γδ T cells may be derived from a subset of CD117- DN thymocytes. Along with other ambiguities, this suggests that T cell development may not be as straightforward as previously thought. To better understand early T cell development, particularly the heterogeneity of DN1 thymocytes, we performed a single cell RNA sequence (scRNAseq) of mouse DN and γδ thymocytes and show that the various DN stages indeed comprise a transcriptionally diverse subpopulations of cells. We also show that multiple subpopulations of DN1 thymocytes exhibit preferential development towards the γδ lineage. Furthermore, specific γδ-primed DN1 subpopulations preferentially develop into IL-17 or IFNγ-producing γδ T cells. We show that DN1 subpopulations that only give rise to IL-17-producing γδ T cells already express many of the transcription factors associated with type 17 immune cell responses, while the DN1 subpopulations that can give rise to IFNγ-producing γδ T cell already express transcription factors associated with type 1 immune cell responses.

Higher well-being individuals are more receptive to cultivated meat: An investigation of their reasoning for consuming cultivated meat.

It is evident that over-consumption of meat can contribute to the emission of hazardous greenhouse gases. One viable way to address such climate impact is to make people become more aware of more sustainable diet options, such as cultivated meat. However, it is challenging to instigate change in people's meat-eating habit, and empirical works have been examining the psychological factors that are related to consumers' willingness to consume cultivated meat. Research has suggested that psychological well-being can play a role in the meaning-making of food consumption, with higher well-being individuals showing more recognition of other sociocultural benefits of consuming food beyond just fulfilling their sustenance needs. As existing works have yet to understand the link between well-being and consumption of novel foods, the current research set out to fill this gap by examining the relationship between people's psychological well-being and their willingness to consume cultivated meat via different reasons (mediators) for consuming cultivated meat. We recruited a representative sample of 948 adults in Singapore to complete an online survey. The study offered the first evidence that there is a positive relationship between people's psychological well-being and their willingness to consume cultivated meat. Further, results revealed that their higher willingness can be motivated by the perception that cultivated meat is as healthy and nutritious, as safe as, and has the same sensory quality as real meat, and is beneficial to the society. This investigation adds to the growing literature on consumer acceptance of cultivated meat by showing the novel finding that well-being and receptivity to cultivated meat is positively linked, and such a positive link can be explained by people's better recognition of the prospective benefits offered by this alternative food.

MYL9 deficiency is neonatal lethal in mice due to abnormalities in the lung and the muscularis propria of the bladder and intestine.

Class II myosin complexes are responsible for muscle contraction as well as other non-sarcomeric contractile functions in cells. Myosin heavy chain molecules form the core of these structures, while light chain molecules regulate their stability and function. MYL9 is a light chain isoform that is thought to regulate non-sarcomeric myosin. However, whether this in only in specific cell types or in all cells remains unclear. To address this, we generated MYL9 deficient mice. These mice die soon after birth with abnormalities in multiple organs. All mice exhibited a distended bladder, shortening of the small intestine and alveolar overdistension in the lung. The Myl9 allele in these mice included a LacZ reporter knockin that allowed for mapping of Myl9 gene expression. Using this reporter, we show that MYL9 expression is restricted to the muscularis propria of the small intestine and bladder, as well as in the smooth muscle layer of the bronchi in the lung and major bladder vessels in all organs. This suggests that MYL9 is important for the function of smooth muscle cells in these organs. Smooth muscle dysfunction is therefore likely to be the cause of the abnormalities observed in the intestine, bladder and lung of MYL9 deficient mice and the resulting neonatal lethality.

Inhibition of the antigen-presenting ability of dendritic cells by non-structural protein 2 of influenza A virus.

Influenza A viruses (IAV), including human IAV and avian IAV (H9N2 subtype), are recurring of influenza outbreaks worldwide in a wide range of mammalian and avian species. Dendritic cells (DCs) are specialised antigen presenting cells. Although DCs can take up IAV and transmit it to other cells, it still unclear why DCs do not effectively present IAV antigens. In this study, we found that Non-structural protein 2 (NS2) of IAV inhibited the maturation and antigen-presenting ability of DCs. We then examined a potential involvement of microRNAs (miRNAs). Analyses of avian DCs stimulated with avian IAV identified 9 upregulated and 10 downregulated miRNAs. However, nearly none microRNA has been significantly altered by NS2 stimulation. Moreover, we found that NS2 binds to exportin 5 (Xpo5), which inhibited miRNA biogenesis. Thus, hijacking of the miRNA biogenesis pathway appears to be one mechanism by which NS2 impairs antigen presentation. Furthermore, we found that NS2 directly interacts with interferon regulatory factor 3, which also inhibits the antigen-presenting ability of DCs. These results thus indicate that NS2-mediated impairment of antigen presentation by DCs might be a mechanism that contributes to the prevalence of the influenza virus.

A cross-country investigation of social image motivation and acceptance of lab-grown meat in Singapore and the United States.

This research has three goals. First, it sets out to compare consumer acceptance of lab-grown meat in the U.S. and in Singapore. Second, it seeks to explain the difference in Americans' and Singaporeans' acceptance of lab-grown meat by examining their eating motivations. Specifically, we focused on social image motivations - the motivations to present oneself positively in social contexts. Third, this study also aims to assess if exposure to information about lab-grown meat communicated by celebrity versus expert social media influencers (SMIs) can impact people's acceptance of lab-grown meat products. Our analysis showed that Singaporean participants had greater acceptance of lab-grown meat compared to their American counterparts, and this cultural difference was explained by Singaporeans' stronger social image eating motivations. In other words, cross-country differences in motivation to eat for a favorable social image can explain differences in consumer acceptance of lab-grown meat. The Singaporean cultural trait of kiasuism, which is exemplified by the fear of losing out or being left behind, may explain Singaporeans' motivation to project an image of being 'trailblazers' (vis-a-vis other nationalities) by expressing a higher acceptance of novel foods such as lab-grown meat. Results also revealed that the information about lab-grown meat being communicated by a celebrity or an expert SMI did not make a difference in participants' acceptance of lab-grown meat in both countries. Together, this research suggests an interesting implication that novel food industries and marketers can promote product branding by boosting media coverage (including online social media) of their lab-grown products' 'firsts' (e.g., the first production line in the world, the first technological breakthrough), especially in markets with high social image concerns.

DROSHA but not DICER is required for human haematopoietic stem cell function.

OBJECTIVES: DROSHA and DICER have central roles in the biogenesis of microRNAs (miRNAs). However, we previously showed that in the murine system, DROSHA has an alternate function where it directly recognises and cleaves protein-coding messenger (m)RNAs and this is critical for safeguarding the pluripotency of haematopoietic stem cells (HSCs). Maintenance of murine HSC function is dependent on DROSHA-mediated cleavage of two mRNAs, Myl9 and Todr1. The goal of this study is to determine whether this pathway is conserved in human HSCs. METHODS: DROSHA and DICER were knocked down in human cord blood CD34+ HSCs with short hairpin RNAs. The function of HSCs was analysed in vitro and in humanised mice. Analysis of mRNA cleavage was performed by capture of 5' phosphorylated RNAs. RESULTS: Consistent with murine HSCs, DROSHA knockdown impaired the differentiation of human HSCs in vitro and engraftment into humanised mice, whereas DICER knockdown had no impact. DROSHA cleaves the MYL9 mRNA in human HSCs and DROSHA deficiency resulted in the accumulation of the mRNA. However, ectopic expression of MYL9 did not impair human HSC function. We were unable to identify a human homolog of Todr1. CONCLUSION: A miRNA-independent function of DROSHA is critical for the function of human HSCs. DROSHA directly recognises and degrades mRNAs in humans HSCs. However, unlike in murine HSCs, the degradation of the MYL9 mRNA alone is not critical for human HSC function. Therefore, DROSHA must be inhibiting other targets and/or has another miRNA-independent function that is essential for safeguarding the pluripotency of human HSCs.

A Retrospective Analysis of Non-Sharps-Related Injuries in a Dental School.

OBJECTIVES: Workplace non-sharps injuries are a common occurrence in a dental school setting. In dentistry, the importance of preventing non-sharps injuries is often overlooked due to emphasis on sharps-related injuries. The aim of this research was to analyse the incidences of non-sharps injuries over an 11-year period in a dental school to identify trends and the possible causative factors for these injuries. METHODS: Injury reports lodged with the University of Queensland Workplace Health and Safety databases between 2009 and 2019 were categorised and analysed. RESULTS: Of 1156 incidents reported, 35.7% (n = 413) were non-sharps injuries, and the most common type of non-sharps injury was general incidents (48.4%, n = 200). The most common body site for injury was the hands (19.4%, n = 80), and the most common location where an injury occurred was in clinical patient care (53.8%, n = 222). The personnel type most at risk of a non-sharps injury fluctuated between students and staff throughout the study period. CONCLUSIONS: All personnel, including students and staff, are at similar risk of experiencing a non-sharps injury within a dental school setting. The equipment and facilities of a dental clinic as well as the level of adherence to safe working procedures are contributing factors. Continuous quality improvement is essential for minimising these injuries.

Expression of the miR-17~92a cluster of microRNAs by regulatory T cells controls blood glucose homeostasis.

Regulatory T cells (Tregs) are a specialized immune cell type that play important roles in regulating immune responses. However, those found in adipose tissue, particularly visceral adipose tissue (VAT), have also been shown to exert metabolic regulatory functions. This study investigated the requirement of the miR-17~92a cluster of microRNAs in VAT Tregs and the impact on blood glucose. This cluster of microRNAs is one that we previously showed to be important for the fitness of Tregs found in secondary lymphoid organs. It was found that male mice with Treg-specific miR-17~92a deficiency are resistant to impaired glucose tolerance induced by a high-fat diet. However, high-fat feeding still impaired glucose tolerance in female mice with Treg-specific miR-17~92a deficiency. There was an increase in KLRG1- naïve Tregs and a loss of KLRG1+ terminally differentiated Tregs in the VAT of Treg-specific miR-17~92a-deficient male mice but not in female mice. The protection of male mice from high-fat feeding was also associated with increased interleukin-10 and reduced interferonγ expression by conventional CD4+ T cells and reduced interleukin-2 expression by both CD4+ and CD8+ T cells in the VAT. Together this suggests that expression of miR-17~92a by VAT Tregs regulates the effector phenotype of conventional T cells and in turn the metabolic function of adipose tissue and blood glucose homeostasis.

Single-Cell RNA Sequencing Approaches for Tracing T Cell Development.

T cell development occurs in the thymus, where uncommitted progenitors are directed into a range of sublineages with distinct functions. The goal is to generate a TCR repertoire diverse enough to recognize potential pathogens while remaining tolerant of self. Decades of intensive research have characterized the transcriptional programs controlling critical differentiation checkpoints at the population level. However, greater precision regarding how and when these programs orchestrate differentiation at the single-cell level is required. Single-cell RNA sequencing approaches are now being brought to bear on this question, to track the identity of cells and analyze their gene expression programs at a resolution not previously possible. In this review, we discuss recent advances in the application of these technologies that have the potential to yield unprecedented insight to T cell development.

Do Patients With Diabetes Have Poorer Improvements in Patient-Reported Outcomes After Total Knee Arthroplasty?

BACKGROUND: Diabetes is one of the most common comorbidities in patients undergoing total knee arthroplasty (TKA) for osteoarthritis. However, the evidence remains unclear on how it affects patient-reported outcome measures after TKA. METHODS: We reviewed prospectively collected data of 2840 patients who underwent primary unilateral TKA between 2008 and 2018, of which 716 (25.2%) had diabetes. All patients had their HbA1c measured within 1 month before surgery, and only well-controlled diabetics (HbA1c <8.0%) were allowed to proceed with surgery. Patient demographics and comorbidities were recorded, and multiple regression was performed to evaluate the impact of diabetes on improvements in patient-reported outcome measures (Short Form 36 (SF-36), Western Ontario and McMaster University Osteoarthritis Index (WOMAC), Knee Society Score (KSS)) and knee range of motion (ROM). RESULTS: Compared with nondiabetics, patients with diabetes were more likely to possess a higher body mass index (P-value <.001), more comorbidities (P-value <.001), and poorer preoperative SF-36 Physical Component Summary (PCS) (P-value <.001), WOMAC (P-value = .002), KSS-function (P-value <.001), and knee ROM (P-value <.001). Multiple regression showed that diabetic patients experienced marginally poorer improvements in KSS-knee (-1.22 points, P-value = .025) and knee ROM (-1.67°, P-value = .013) than nondiabetics. However, there were no significant differences in improvements for SF-36 PCS (P-value = .163), Mental Component Summary (P-value = .954), WOMAC (P-value = .815), and KSS-function (P-value = .866). CONCLUSION: Patients with well-controlled diabetes (HbA1c <8.0%) can expect similar improvements in general health and osteoarthritis outcomes (SF-36 PCS and Mental Component Summary, WOMAC, and KSS-function) compared with nondiabetics after TKA. Despite having marginally poorer improvements in knee-specific outcomes (KSS-knee and knee ROM), these differences are unlikely to be clinically significant.

A comparison of alternative mRNA splicing in the CD4 and CD8 T cell lineages.

T cells can be subdivided into a number of different subsets that are defined by their distinct functions. While the specialization of different T cell subsets is partly achieved by the expression of specific genes, the overall transcriptional profiles of all T cells appear very similar. Alternative mRNA splicing is a mechanism that facilitates greater transcript/protein diversity from a limited number of genes, which may contribute to the functional specialization of distinct T cell subsets. In this study we employ a combination of short-read and long-read sequencing technologies to compare alternative mRNA splicing between the CD4 and CD8 T cell lineages. While long-read technology was effective at assembling full-length alternatively spliced transcripts, the low sequencing depth did not facilitate accurate quantitation. On the other hand, short-read technology was ineffective at assembling full-length transcripts but was highly accurate for quantifying expression. We show that integrating long-read and short-read data together achieves a more complete view of transcriptomic diversity. We found that while the overall usage of transcript isoforms was very similar between the CD4 and CD8 lineages, there were numerous alternative spliced mRNA isoforms that were preferentially used by one lineage over the other. These alternative spliced isoforms included ones with different exon usage, exon exclusion or intron inclusion, all of which are expected to significantly alter the protein sequence.

Regulating gene expression in animals through RNA endonucleolytic cleavage.

The expression of any gene must be precisely controlled for appropriate function. This expression can be controlled at various levels. This includes epigenetic regulation through DNA methylation or histone modifications. At the posttranscriptional level, regulation can be via alternative splicing or controlling messenger RNA (mRNA) stability. RNA cleavage is one way to control mRNA stability. For example, microRNA (miRNA)-induced mRNA cleavage has long been recognised in plants. RNA cleavage also appears to be widespread in other kingdoms of life, and it is now clear that mRNA cleavage plays critical functions in animals. Although miRNA-induced mRNA cleavage can occur in animals, it is not a widespread mechanism. Instead, mRNA cleavage can be induced by a range of other mechanisms, including by endogenous short inhibitory RNAs (endo-siRNAs), as well as the Ribonuclease III (RNase III) enzymes Drosha and Dicer. In addition, RNA cleavage induced by endo-siRNAs and PIWI-interacting RNAs (piRNAs) is important for genome defence against transposons. Moreover, several RNase has been identified as important antiviral mediators. In this review, we will discuss these various RNA endonucleolytic cleavage mechanisms utilised by animals to regulate the expression of genes and as a defence against retrotransposons and viral infection.

Endothelial colony forming cells from human umbilical cord blood improved severe erectile dysfunction in obese type II diabetic rats.

AIM: To investigate the effect of intracavernous injection of human umbilical cord blood derived endothelial colony forming cells (HUCB ECFCs) on erectile dysfunction (ED) in Zucker Diabetic Fatty (ZDF) rat model. METHODS: Erectile function was assessed by cavernous nerve electrostimulation in ZDF rats aged 20-28 weeks. Following confirmation of severe ED at the age of 28 weeks, 21 ZDF rats were randomly assigned to three experimental groups: 1 million ECFCs, 2 million ECFCs, and phosphate buffered saline (PBS). Four weeks after intracavernous injection, the efficacy of ECFCs was quantified by intracavernous pressure (ICP) measurement, Masson's trichrome staining, immunohistologic and immunoblot analyses and TUNEL assay. KEY FINDINGS: Intracavernous ECFC administration improved ICP in a dose-dependent manner in comparison to the age-matched PBS group. Functional improvement in ICP was accompanied by a significant restoration of the cavernosal endothelial and smooth muscle cell content and cavernosal nerve function. The percentage eNOS and nNOS positive cavernosal cells, and their respective protein expression levels and nNOS positive cells in the dorsal penile nerve in 2 million ECFCs treated groups were significantly higher than the PBS group. TUNEL stain quantification showed a significant decrease in cavernosal apoptosis following ECFC treatment. SIGNIFICANCE: The results are expected to provide a scientific basis to further study the clinical application of HUCB ECFCs in ameliorating ED in human. CONCLUSIONS: HUCB ECFCs significantly improved severe ED in ZDF rats through improvement of the nerve and endothelium function and restoration of smooth muscle in the cavernosum by overcoming the cavernosal apoptosis.