Using Text Messaging Surveys in General Practice Research to Engage With People From Low-Income Groups: Multi-Methods Study.

BACKGROUND: SMS text messages through mobile phones are a common means of interpersonal communication. SMS text message surveys are gaining traction in health care and research due to their feasibility and patient acceptability. However, challenges arise in implementing SMS text message surveys, especially when targeting marginalized populations, because of barriers to accessing phones and data as well as communication difficulties. In primary care, traditional surveys (paper-based and online) often face low response rates that are particularly pronounced among disadvantaged groups due to financial limitations, language barriers, and time constraints. OBJECTIVE: This study aimed to investigate the potential of SMS text message-based patient recruitment and surveys within general practices situated in lower socioeconomic areas. This study was nested within the Reducing Alcohol-Harm in General Practice project that aimed to reduce alcohol-related harm through screening in Australian general practice. METHODS: This study follows a 2-step SMS text message data collection process. An initial SMS text message with an online survey link was sent to patients, followed by subsequent surveys every 3 months for consenting participants. Interviews were conducted with the local primary health network organization staff, the participating practice staff, and the clinicians. The qualitative data were analyzed using constructs from the Consolidated Framework for Implementation Research. RESULTS: Out of 6 general practices, 4 were able to send SMS text messages to their patients. The initial SMS text message was sent to 8333 patients and 702 responses (8.2%) were received, most of which were not from a low-income group. This low initial response was in contrast to the improved response rate to the ongoing 3-month SMS text message surveys (55/107, 51.4% at 3 months; 29/67, 43.3% at 6 months; and 44/102, 43.1% at 9 months). We interviewed 4 general practitioners, 4 nurses, and 4 administrative staff from 5 of the different practices. Qualitative data uncovered barriers to engaging marginalized groups including limited smartphone access, limited financial capacity (telephone, internet, and Wi-Fi credit), language barriers, literacy issues, mental health conditions, and physical limitations such as manual dexterity and vision issues. Practice managers and clinicians suggested strategies to overcome these barriers, including using paper-based surveys in trusted spaces, offering assistance during survey completion, and offering honoraria to support participation. CONCLUSIONS: While SMS text message surveys for primary care research may be useful for the broader population, additional efforts are required to ensure the representation and involvement of marginalized groups. More intensive methods such as in-person data collection may be more appropriate to capture the voice of low-income groups in primary care research. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.3399/BJGPO.2021.0037.

Blood-based multivariate methylation risk score for cognitive impairment and dementia.

INTRODUCTION: The established link between DNA methylation and pathophysiology of dementia, along with its potential role as a molecular mediator of lifestyle and environmental influences, positions blood-derived DNA methylation as a promising tool for early dementia risk detection. METHODS: In conjunction with an extensive array of machine learning techniques, we employed whole blood genome-wide DNA methylation data as a surrogate for 14 modifiable and non-modifiable factors in the assessment of dementia risk in independent dementia cohorts. RESULTS: We established a multivariate methylation risk score (MMRS) for identifying mild cognitive impairment cross-sectionally, independent of age and sex (P = 2.0 × 10-3). This score significantly predicted the prospective development of cognitive impairments in independent studies of Alzheimer's disease (hazard ratio for Rey's Auditory Verbal Learning Test (RAVLT)-Learning = 2.47) and Parkinson's disease (hazard ratio for MCI/dementia = 2.59). DISCUSSION: Our work shows the potential of employing blood-derived DNA methylation data in the assessment of dementia risk. HIGHLIGHTS: We used whole blood DNA methylation as a surrogate for 14 dementia risk factors. Created a multivariate methylation risk score for predicting cognitive impairment. Emphasized the role of machine learning and omics data in predicting dementia. The score predicts cognitive impairment development at the population level.

Casein Kinase 1α-A Target for Prostate Cancer Therapy?

The androgen receptor (AR) is a key driver of prostate cancer (PCa) and, as such, current mainstay treatments target this molecule. However, resistance commonly arises to these therapies and, therefore, additional targets must be evaluated to improve patient outcomes. Consequently, alternative approaches for indirectly targeting the AR are sought. AR crosstalk with other signalling pathways, including several protein kinase signalling cascades, has been identified as a potential route to combat therapy resistance. The casein kinase 1 (CK1) family of protein kinases phosphorylate a multitude of substrates, allowing them to regulate a diverse range of pathways from the cell cycle to DNA damage repair. As well as its role in several signalling pathways that are de-regulated in PCa, mutational data suggest its potential to promote prostate carcinogenesis. CK1α is one isoform predicted to regulate AR activity via phosphorylation and has been implicated in the progression of several other cancer types. In this review, we explore how the normal biological function of CK1 is de-regulated in cancer, the impact on signalling pathways and how this contributes towards prostate tumourigenesis, with a particular focus on the CK1α isoform as a novel therapeutic target for PCa.

Direct activation of PI3K in osteoblasts and osteocytes strengthens murine bone through sex-specific actions on cortical surfaces.

Intracellular phosphoinositide 3-kinase (PI3K) signaling is activated by multiple bone-active receptors. Genetic mutations activating PI3K signaling are associated with clinical syndromes of tissue overgrowth in multiple organs, often including the skeleton. While one formation is increased by removing the PI3K inhibitor (phosphatase and TENsin homolog deleted on chromosome 10 (PTEN)), the effect of direct PI3K activation in the osteoblast lineage has not been reported. We introduced a known gain-of-function mutation in Pik3ca, the gene encoding the p110α catalytic subunit of PI3K, in osteocytes and late osteoblasts using the dentin matrix protein-1 Cre (Dmp1Cre) mouse and assessed the skeletal phenotype. Femur shape was grossly normal, but cortical thickness was significantly greater in both male and female Dmp1Cre.Pik3caH1047R mice, leading to almost doubled bone strength at 12 wk of age. Both sexes had smaller marrow areas from 6 wk of age. Female mice also exhibited greater cross-sectional area, which continued to increase until 24 wk of age, resulting in a further increase in bone strength. Although both male and female mice had increased endocortical mineralizing surface, only female mice had increased periosteal mineralizing surface. The bone formed in the Dmp1Cre.Pik3caH1047R mice showed no increase in intracortical remodeling nor any defect in cortical bone consolidation. In contrast, on both endocortical and periosteal surfaces, there was more lamellar bone formation, including highly organized osteocyte networks extending along the entire surface at a greater thickness than in control mice. In conclusion, direct activation of PI3Kα in cells targeted by Dmp1Cre leads to high cortical bone mass and strength with abundant lamellar cortical bone in female and male mice with no increase in intracortical remodeling. This differs from the effect of PTEN deletion in the same cells, suggesting that activating PI3Kα in osteoblasts and osteocytes may be a more suitable target to promote formation of lamellar bone.

Patients with genetic activation of enzymes called phosphoinositide-3 kinase (PI3K) have tissue overgrowth syndromes, where parts of the body become enlarged, sometimes including the skeleton. There are 2 types of mutations that cause this: one that directly activates the PI3K enzyme, and one that removes the normal brake on PI3K signaling (called PTEN). We tested the effect of directly activating a PI3K enzyme specifically in osteoblasts (the cells that form bone) and osteocytes (osteoblasts that make a network inside the bone tissue itself). We found that mice with these mutations had very strong bones with an outer shell that was thicker than usual. In both male and female mice, it became thicker on the inside of the shell, but in female mice it also became thicker on the outside, making the bones even stronger over time. The new bone was well-organized, which likely helped make the increase in bone strength so profound. This is very different to previous studies of mice with the other type of mutation in their bone-forming cells; they had a shell with many large holes (pores). This indicates that directly stimulating PI3K enzyme is more beneficial for bone than removing the PTEN brake.

Realist process evaluation of occupational performance coaching: protocol.

INTRODUCTION: A cluster randomised controlled trial, the Meaning, Agency and Nurturing Autonomy (MANA) study, is underway comparing the effects of occupational performance coaching (OPC) and usual care on the social participation, health and well-being of children with neurodisability and their caregivers. This protocol presents the realist process evaluation which is occurring in parallel with the trial to allow testing and further refinement of OPC programme theory, as represented in its logic model. The aim of this realist evaluation is to examine what works, for whom, in the implementation of OPC with caregivers of children with neurodisability (in particular, Maori and Pasifika) in current service delivery contexts. METHODS AND ANALYSIS: Guided by OPC programme theory and realist evaluation processes, mixed-methods data collected from the MANA study OPC group will be analysed to elucidate when OPC works (outcomes), for whom, how (mechanisms) and under what circumstances (contexts). This will culminate in the synthesis of Intervention-Actor Context-Mechanism-Outcome configurations. Descriptive analyses will be reported for quantitative measures of treatment fidelity (OPC-Fidelity Measure), caregiver emotional response to OPC (Session Rating Scale) preintervention emotional state (Depression Stress and Anxiety Scale) and client outcomes (Canadian Occupational Performance Measure). Reflexive thematic analysis will be undertaken to analyse realist interviews with therapists who implemented OPC above and below fidelity thresholds and culturally focused interviews with clients of Maori or Pasifika ethnicity, informing understanding of the contexts influencing therapists' implementation of OPC with fidelity, and the mechanisms triggered within therapists or caregivers to elicit a response to the intervention. The MANA study trial outcomes will be reported separately. ETHICS AND DISSEMINATION: Ethical approval for this study was granted by the New Zealand Health and Disability Ethics Committee (20/STH/93). In all participating jurisdictions local area approval was obtained, involving a process of local Maori consultation. Results will be disseminated to all participants, and more broadly to clinicians and policy-makers through conference presentations and peer-reviewed journal publications, which will inform decision-making about resourcing and supporting effective delivery of OPC to optimise outcomes for children and caregivers. TRIAL REGISTRATION NUMBER: ACTRN12621000519853.

Developmentally dynamic changes in DNA methylation in the human pancreas.

Development of the human pancreas requires the precise temporal control of gene expression via epigenetic mechanisms and the binding of key transcription factors. We quantified genome-wide patterns of DNA methylation in human fetal pancreatic samples from donors aged 6 to 21 post-conception weeks. We found dramatic changes in DNA methylation across pancreas development, with > 21% of sites characterized as developmental differentially methylated positions (dDMPs) including many annotated to genes associated with monogenic diabetes. An analysis of DNA methylation in postnatal pancreas tissue showed that the dramatic temporal changes in DNA methylation occurring in the developing pancreas are largely limited to the prenatal period. Significant differences in DNA methylation were observed between males and females at a number of autosomal sites, with a small proportion of sites showing sex-specific DNA methylation trajectories across pancreas development. Pancreas dDMPs were not distributed equally across the genome and were depleted in regulatory domains characterized by open chromatin and the binding of known pancreatic development transcription factors. Finally, we compared our pancreas dDMPs to previous findings from the human brain, identifying evidence for tissue-specific developmental changes in DNA methylation. This study represents the first systematic exploration of DNA methylation patterns during human fetal pancreas development and confirms the prenatal period as a time of major epigenomic plasticity.

Comparing stimulus preference and response force in a conjugate preparation: A replication with auditory stimulation.

This study examined a conjugate approach for evaluating auditory stimulus preference for 81 participants using force as a continuous response dimension. First, the researchers used a verbal preference assessment to evaluate each participant's preference for listening to five genres of music. This process identified high-preference and low-preference music for each participant. Thereafter, the researchers exposed each participant to the five music genres in a randomized order while using a hand dynamometer to measure their response force to increase the auditory clarity of the music. The results indicate (a) 63% of the participants' high-preference music genres corresponded to the genre for which they exerted the highest mean force and (b) most participants' low-preference music genres corresponded to the genre for which they exerted the lowest mean force. These findings are consistent with those from Davis et al. (2021) and further support using conjugate preparations for measuring the relative value of some stimulus events.

Cooperative Conformational Transitions Underpin the Activation Heat Capacity in the Temperature Dependence of Enzyme Catalysis.

Many enzymes display non-Arrhenius behavior with curved Arrhenius plots in the absence of denaturation. There has been significant debate about the origin of this behavior and recently the role of the activation heat capacity (ΔCP⧧) has been widely discussed. If enzyme-catalyzed reactions occur with appreciable negative values of ΔCP⧧ (arising from narrowing of the conformational space along the reaction coordinate), then curved Arrhenius plots are a consequence. To investigate these phenomena in detail, we have collected high precision temperature-rate data over a wide temperature interval for a model glycosidase enzyme MalL, and a series of mutants that change the temperature-dependence of the enzyme-catalyzed rate. We use these data to test a range of models including macromolecular rate theory (MMRT) and an equilibrium model. In addition, we have performed extensive molecular dynamics (MD) simulations to characterize the conformational landscape traversed by MalL in the enzyme-substrate complex and an enzyme-transition state complex. We have crystallized the enzyme in a transition state-like conformation in the absence of a ligand and determined an X-ray crystal structure at very high resolution (1.10 Å). We show (using simulation) that this enzyme-transition state conformation has a more restricted conformational landscape than the wildtype enzyme. We coin the term "transition state-like conformation (TLC)" to apply to this state of the enzyme. Together, these results imply a cooperative conformational transition between an enzyme-substrate conformation (ES) and a transition-state-like conformation (TLC) that precedes the chemical step. We present a two-state model as an extension of MMRT (MMRT-2S) that describes the data along with a convenient approximation with linear temperature dependence of the activation heat capacity (MMRT-1L) that can be used where fewer data points are available. Our model rationalizes disparate behavior seen for MalL and previous results for a thermophilic alcohol dehydrogenase and is consistent with a raft of data for other enzymes. Our model can be used to characterize the conformational changes required for enzyme catalysis and provides insights into the role of cooperative conformational changes in transition state stabilization that are accompanied by changes in heat capacity for the system along the reaction coordinate. TLCs are likely to be of wide importance in understanding the temperature dependence of enzyme activity and other aspects of enzyme catalysis.

Loss of maternal calcitriol reversibly alters early offspring growth and skeletal development in mice.

Ablation of Cyp27b1 eliminates calcitriol but does not disturb fetal mineral homeostasis or skeletal development. However, independent of fetal genotypes, maternal loss of Cyp27b1 altered fetal mineral and hormonal levels compared to offspring of WT dams. We hypothesized that these maternal influences would alter postnatal skeletal development. Cyp27b1 null and WT females were mated to bear only Cyp27b1+/- offspring. Forty-eight hours after birth, pups were cross-fostered to dams of the same or opposite genotype that bore them. Maternal and offspring samples were collected on days 21 (weaning) and 42. Offspring measurements included minerals and hormones, BMC by DXA, ash weight and mineral content, gene expression, 3-point bending tests, and microCT. Maternal lactational behavior was evaluated. Milk was analyzed for nutritional content. At day 21, offspring fostered by nulls, independent of birth dam, had ~20% lower weight, BMC, ash weight, and ash calcium than pups fostered by WT dams. Adjustment for body weight accounted for the lower BMC but not the lower ash weight and ash calcium. Hormones and serum/urine minerals did not differ across offspring groups. Offspring fostered by nulls had shorter femurs and lower cortical thickness, mean polar moment of inertia, cortical area, trabecular bone volume, and trabecular number. Dam lactational behaviors and milk nutritional content did not differ between groups. At day 42, body weight, ash weight, lengths, BMC, and tibial bone strength were no longer different between pups fostered by null vs WT dams. In summary, pups fostered by Cyp27b1 nulls, regardless of birth dam, have proportionately smaller skeletons at 21 d, impaired microstructure, but normal mineral homeostasis. The skeletal effects are largely recovered by day 42 (3 wk after weaning). In conclusion, maternal loss of calcitriol impairs early postnatal cortical bone growth and trabecular bone mass, but affected offspring catch up after weaning.

Quantifying the proportion of different cell types in the human cortex using DNA methylation profiles.

BACKGROUND: Due to interindividual variation in the cellular composition of the human cortex, it is essential that covariates that capture these differences are included in epigenome-wide association studies using bulk tissue. As experimentally derived cell counts are often unavailable, computational solutions have been adopted to estimate the proportion of different cell types using DNA methylation data. Here, we validate and profile the use of an expanded reference DNA methylation dataset incorporating two neuronal and three glial cell subtypes for quantifying the cellular composition of the human cortex. RESULTS: We tested eight reference panels containing different combinations of neuronal- and glial cell types and characterised their performance in deconvoluting cell proportions from computationally reconstructed or empirically derived human cortex DNA methylation data. Our analyses demonstrate that while these novel brain deconvolution models produce accurate estimates of cellular proportions from profiles generated on postnatal human cortex samples, they are not appropriate for the use in prenatal cortex or cerebellum tissue samples. Applying our models to an extensive collection of empirical datasets, we show that glial cells are twice as abundant as neuronal cells in the human cortex and identify significant associations between increased Alzheimer's disease neuropathology and the proportion of specific cell types including a decrease in NeuNNeg/SOX10Neg nuclei and an increase of NeuNNeg/SOX10Pos nuclei. CONCLUSIONS: Our novel deconvolution models produce accurate estimates for cell proportions in the human cortex. These models are available as a resource to the community enabling the control of cellular heterogeneity in epigenetic studies of brain disorders performed on bulk cortex tissue.

Periods Shouldn't Bring Any Adolescents' World to a Full Stop. Period. An Online Survey of Adolescents' Experience of Menstruation.

STUDY OBJECTIVE: Few studies have explored what specific outcome measures contained in assessment tools for period and pelvic pain are most relevant to adolescents. Co-design is a valuable method of ensuring input from those with lived experience. The Longitudinal Study of Teenagers with Endometriosis Periods and Pelvic Pain in Australia (LongSTEPPP) Co-Design Periods Survey comprised an anonymous online survey of adolescents' experience of menstruation to inform patient-reported outcome measures for the larger 5-year project. METHODS: Adolescents aged 12-18 years whose periods had commenced at least 3 months previously and with demonstrated capacity to consent were invited to participate in an online survey. Recruitment was primarily via social media channels. RESULTS: Of the 1811 adolescents who participated, 85% reported that periods had a "moderate" or greater impact on their life. Pain (90.7%), heavy flow (56.2%), and worry about leakage (49%) were common reasons for missed activities. Menstrual symptoms were wide-ranging and included cramping, nausea, poor energy, and impacts on mood. When asked where adolescents sought assistance with their periods, 39.8% had seen their general practitioner, 21.3% their school nurse, and almost 1 in 10 had consulted a mental health practitioner (9.3%). To manage menstrual symptoms, heat packs (66.0%), over-the-counter medications (55.8%), and prescription medications (28.6%) were used. CONCLUSION: We found a lack of menstrual health awareness in adolescents. Periods had a significant effect on their lives, and adolescents commonly missed activities. In managing menstruation, a wide range of practitioners were consulted. Nearly a third were prescribed medication to manage their periods. These findings have directed the longitudinal study as to how best to capture outcome measures that reflect the impact of periods on adolescents.

Congenital anomalies of the kidney and urinary tract: antenatal diagnosis, management and counselling of families.

Congenital anomalies of the kidney and urinary tract are collectively one of the most commonly diagnosed antenatal conditions. Clinicians have several tools available to diagnose anomalies, including imaging, biomarkers, family history and genetic studies. In certain cases, antenatal interventions such as vesico-amniotic shunting may be considered to improve postnatal outcomes.Congenital kidney anomalies detected antenatally can vary in clinical significance from almost no impact postnatally to significant morbidity and perinatal mortality. Prognosis broadly depends on kidney size, structure and amount of amniotic fluid, alongside genetics and family history, and progression on subsequent scans. It is important to counsel parents appropriately using a parent-focused and personalised approach. The use of a multidisciplinary team should always be considered.

Inherited non-FGF23-mediated phosphaturic disorders: A kidney-centric review.

Phosphate is freely filtered by the glomerulus and reabsorbed exclusively in the proximal tubule by two key transporters, NaPiIIA and NaPiIIC, encoded by SLC34A1 and SLC34A3, respectively. Regulation of these transporters occurs primarily through the hormone FGF23 and, to a lesser degree, PTH. Consequently, inherited non-FGF23 mediated phosphaturic disorders are due to generalised proximal tubular dysfunction, loss-of-function variants in SLC34A1 or SLC34A3 or excess PTH signalling. The corresponding disorders are Renal Fanconi Syndrome, Infantile Hypercalcaemia type 2, Hereditary Hypophosphataemic Rickets with Hypercalciuria and Familial Hyperparathyroidism. Several inherited forms of Fanconi renotubular syndrome (FRTS) have also been described with the underlying genes encoding for GATM, EHHADH, HNF4A and NDUFAF6. Here, we will review their pathophysiology, clinical manifestations and the implications for treatment from a kidney-centric perspective, focussing on those disorders caused by dysfunction of renal phosphate transporters. Moreover, we will highlight specific genetic aspects, as the availability of large population genetic databases has raised doubts about some of the originally proposed gene-disease associations concerning phosphate transporters or their associated proteins.

Design and assembly of the 117-kb Phaeodactylum tricornutum chloroplast genome.

There is growing impetus to expand the repertoire of chassis available to synthetic biologists. Chloroplast genomes present an interesting alternative for engineering photosynthetic eukaryotes; however, development of the chloroplast as a synthetic biology chassis has been limited by a lack of efficient techniques for whole-genome cloning and engineering. Here, we demonstrate two approaches for cloning the 117-kb Phaeodactylum tricornutum chloroplast genome that have 90% to 100% efficiency when screening as few as 10 yeast (Saccharomyces cerevisiae) colonies following yeast assembly. The first method reconstitutes the genome from PCR-amplified fragments, whereas the second method involves precloning these fragments into individual plasmids from which they can later be released. In both cases, overlapping fragments of the chloroplast genome and a cloning vector are homologously recombined into a singular contig through yeast assembly. The cloned chloroplast genome can be stably maintained and propagated within Escherichia coli, which provides an exciting opportunity for engineering a delivery mechanism for bringing DNA directly to the algal chloroplast. Also, one of the cloned genomes was designed to contain a single SapI site within the yeast URA3 (coding for orotidine-5'-phosphate decarboxylase) open-reading frame, which can be used to linearize the genome and integrate designer cassettes via golden-gate cloning or further iterations of yeast assembly. The methods presented here could be extrapolated to other species-particularly those with a similar chloroplast genome size and architecture (e.g. Thalassiosira pseudonana).

Enhancing operative documentation of emergency laparotomy: a systematic review and development of a synoptic reporting template.

INTRODUCTION: Currently, operative reports are narrative and often handwritten, making interpretation difficult and potentially omitting key steps of the procedure. This study undertook a systematic review to determine the current availability of synoptic operative reporting and develop a synoptic operative record template for emergency laparotomy (EL). METHODS: A PROSPERO registered study from January 1st, 2012, to December 31st, 2022, was conducted using PubMed, Scopus, and Web of Science databases in February 2023. KEYWORDS: emergency laparotomy AND operation notes OR operative notes OR documentation OR report OR pro forma OR narrative OR synoptic OR digital OR audio-visual. Studies on paediatric or pregnant patients, systematic reviews, meta-analyses, case reports, editorial comments, and letters were excluded. A synoptic operative record was designed to include key standards in the documentation, as suggested by the Colleges of Surgeons. RESULTS: The literature search yielded 4687 articles, and no relevant published articles were found. A detailed synoptic template was developed, which included 111 fields related to patient demographics, operative findings, interventions, and documentation of key variables associated with patient outcomes. 11 were text boxes, two were related to digital audio-visual uploads, and three facilitated the digital scoring/grading of findings. CONCLUSION: This systematic review identified a limited number of publications reporting synoptic operative reporting, and none related to emergency laparotomy. This novel operative template provides a platform for clear documentation of the surgery performed during emergency laparotomy, potentially facilitating data analysis, resident training, and research, in turn leading to a better understanding of patient outcomes.

A Bourdieusian Latent Class Analysis of Cultural, Arts, Heritage and Sports Activities in the UK Representative Understanding Society Dataset.

To Bourdieu, interaction with culture has symbolic power and drives the manifestation of social stratification. Many have adapted his theory and methodology, developing new models of cultural engagement. Here, to further integrate these theoretical and methodological approaches, Bourdieu's tools were used to operationalise and interpret a Latent Class Analysis of cultural engagement in the Understanding Society dataset. Six classes of increasing engagement were established, and were increasingly correlated with youth, capital and social advantage. However, some qualitative differences in engagement were also seen. The classes also varied by which characteristics correlated with membership. For example, economic capital was associated with sports engagement, while advantaged social position was associated with broad-scale engagement. Overall, this analysis combined Bourdieusian theory with contemporary methodology in the largest representative UK dataset and highlights the broader relevance of cultural engagement patterns in indicating (and possibly generating) status, identity, capital and social position.

CDC20 Is Regulated by the Histone Methyltransferase, KMT5A, in Castration-Resistant Prostate Cancer.

The methyltransferase KMT5A has been proposed as an oncogene in prostate cancer and therefore represents a putative therapeutic target. To confirm this hypothesis, we have performed a microarray study on a prostate cancer cell line model of androgen independence following KMT5A knockdown in the presence of the transcriptionally active androgen receptor (AR) to understand which genes and cellular processes are regulated by KMT5A in the presence of an active AR. We observed that 301 genes were down-regulated whilst 408 were up-regulated when KMT5A expression was reduced. KEGG pathway and gene ontology analysis revealed that apoptosis and DNA damage signalling were up-regulated in response to KMT5A knockdown whilst protein folding and RNA splicing were down-regulated. Under these conditions, the top non-AR regulated gene was found to be CDC20, a key regulator of the spindle assembly checkpoint with an oncogenic role in several cancer types. Further investigation revealed that KMT5A regulates CDC20 in a methyltransferase-dependent manner to modulate histone H4K20 methylation within its promoter region and indirectly via the p53 signalling pathway. A positive correlation between KMT5A and CDC20 expression was also observed in clinical prostate cancer samples, further supporting this association. Therefore, we conclude that KMT5A is a valid therapeutic target for the treatment of prostate cancer and CDC20 could potentially be utilised as a biomarker for effective therapeutic targeting.

Evidence for a short-lived resonance state in enzyme catalysis via rate-equation convolution.

At the cellular level, all biological function relies on enzymes to provide catalytic acceleration of essential biochemical processes driving cellular metabolism. The enzyme is presumed to lower the activation energy barrier separating reactants from products, but the precise mechanism remains unresolved. Here we examine the temperature dependence of the enzyme-catalyzed dissociation of p-nitrophenyl-α-D-glucopyranoside (pNPG), a chromogenic analog for maltose, isomaltose, and sucrose disaccharide sugars, into p-nitrophenol (pNP) and glucose (monosaccharide). The enzymes of interest are the wild type and mutant forms of glucosidase MalL produced by the probiotic bacterium Bacillus subtilis. The per-enzyme production rates k(T) for the pNPG→ glucose reaction all show a characteristic temperature profile with an Arrhenius-like (approximately exponential) slow acceleration at low temperatures, rising through a point of inflexion to reach a maximum, then turning over to decline steeply towards zero production at high temperatures. This asymmetric profile is found to be well fitted by convolving an exponential growth function f(T) with a Gaussian temperature distribution g(T) to produce an exponentially modified Gaussian function h(T). To give a physical interpretation of the convolution components, we make the temperature mapping Θ≡T_{ref}-T where T_{ref} marks the temperature at which a given mutant becomes fully denatured (unfolded) and therefore inactive, then convert the convolution components to probability density functions which obey the convolution theorem of statistics. Working in Θ space, we identify f(Θ) as the density function for an Arrhenius-like transition from ground-state A to metastable-state B, and g(Θ) as the Gaussian distribution of offset-temperature fluctuations for the metastable state. By mapping the standard thermodynamic relations for temperature and energy fluctuations to the enzyme frame of reference, we are able to derive an expression for the lifetime for the metastable B state. For the 15 enzyme experiments, we obtain a mean value 〈Δt〉≳(29.0±1.3)×10^{-15}s, in remarkably good agreement with the ∼30-fs estimate for the period of glycosidic bond oscillations extracted from published infrared spectroscopy. We suggest that the metastable B state provides a low-energy target that has the effect of lowering the activation energy barrier by presenting an alternative axis for the reaction coordinate.

Expression of non-phosphorylatable S5A-L-plastin exerts phenotypes distinct from L-plastin deficiency during podosome formation and phagocytosis.

Introduction: The actin cytoskeleton remodels to enable diverse processes essential to immunity, such as cell adhesion, migration and phagocytosis. A panoply of actin-binding proteins regulate these rapid rearrangements to induce actin-based shape changes and to generate force. L-plastin (LPL) is a leukocyte-specific, actin-bundling protein that is regulated in part by phosphorylation of the Ser-5 residue. LPL deficiency in macrophages impairs motility, but not phagocytosis; we recently found that expression of LPL in which the S5 residue is converted to a non-phosphorylatable alanine (S5A-LPL) resulted in diminished phagocytosis, but unimpaired motility. Methods: To provide mechanistic insight into these findings, we now compare the formation of podosomes (an adhesive structure) and phagosomes in alveolar macrophages derived from wild-type (WT), LPL-deficient, or S5A-LPL mice. Both podosomes and phagosomes require rapid remodeling of actin, and both are force-transmitting. Actin rearrangement, force generation, and signaling rely upon recruitment of many actin-binding proteins, including the adaptor protein vinculin and the integrin-associated kinase Pyk2. Prior work suggested that vinculin localization to podosomes was independent of LPL, while Pyk2 was displaced by LPL deficiency. We therefore chose to compare vinculin and Pyk2 co-localization with F-actin at sites of adhesion of phagocytosis in AMs derived from WT, S5A-LPL or LPL-/- mice, using Airyscan confocal microscopy. Results: As described previously, podosome stability was significantly disrupted by LPL deficiency. In contrast, LPL was dispensable for phagocytosis and was not recruited to phagosomes. Recruitment of vinculin to sites of phagocytosis was significantly enhanced in cells lacking LPL. Expression of S5A-LPL impeded phagocytosis, with reduced appearance of ingested bacteria-vinculin aggregates. Discussion: Our systematic analysis of the regulation of LPL during podosome vs. phagosome formation illuminates essential remodeling of actin during key immune processes.

Mechanisms of host manipulation by Neisseria gonorrhoeae.

Neisseria gonorrhoeae (also known as gonococcus) has been causing gonorrhoea in humans since ancient Egyptian times. Today, global gonorrhoea infections are rising at an alarming rate, in concert with an increasing number of antimicrobial-resistant strains. The gonococcus has concurrently evolved several intricate mechanisms that promote pathogenesis by evading both host immunity and defeating common therapeutic interventions. Central to these adaptations is the ability of the gonococcus to manipulate various host microenvironments upon infection. For example, the gonococcus can survive within neutrophils through direct regulation of both the oxidative burst response and maturation of the phagosome; a concerning trait given the important role neutrophils have in defending against invading pathogens. Hence, a detailed understanding of how N. gonorrhoeae exploits the human host to establish and maintain infection is crucial for combating this pathogen. This review summarizes the mechanisms behind host manipulation, with a central focus on the exploitation of host epithelial cell signaling to promote colonization and invasion of the epithelial lining, the modulation of the host immune response to evade both innate and adaptive defenses, and the manipulation of host cell death pathways to both assist colonization and combat antimicrobial activities of innate immune cells. Collectively, these pathways act in concert to enable N. gonorrhoeae to colonize and invade a wide array of host tissues, both establishing and disseminating gonococcal infection.