The expanding amyloid family: Structure, stability, function, and pathogenesis.

The hidden world of amyloid biology has suddenly snapped into atomic-level focus, revealing over 80 amyloid protein fibrils, both pathogenic and functional. Unlike globular proteins, amyloid proteins flatten and stack into unbranched fibrils. Stranger still, a single protein sequence can adopt wildly different two-dimensional conformations, yielding distinct fibril polymorphs. Thus, an amyloid protein may define distinct diseases depending on its conformation. At the heart of this conformational variability lies structural frustrations. In functional amyloids, evolution tunes frustration levels to achieve either stability or sensitivity according to the fibril's biological function, accounting for the vast versatility of the amyloid fibril scaffold.

Organ-level quorum sensing directs regeneration in hair stem cell populations.

Coordinated organ behavior is crucial for an effective response to environmental stimuli. By studying regeneration of hair follicles in response to patterned hair plucking, we demonstrate that organ-level quorum sensing allows coordinated responses to skin injury. Plucking hair at different densities leads to a regeneration of up to five times more neighboring, unplucked resting hairs, indicating activation of a collective decision-making process. Through data modeling, the range of the quorum signal was estimated to be on the order of 1 mm, greater than expected for a diffusible molecular cue. Molecular and genetic analysis uncovered a two-step mechanism, where release of CCL2 from injured hairs leads to recruitment of TNF-α-secreting macrophages, which accumulate and signal to both plucked and unplucked follicles. By coupling immune response with regeneration, this mechanism allows skin to respond predictively to distress, disregarding mild injury, while meeting stronger injury with full-scale cooperative activation of stem cells.

Modeling the emergence of viral resistance for SARS-CoV-2 during treatment with an anti-spike monoclonal antibody.

To mitigate the loss of lives during the COVID-19 pandemic, emergency use authorization was given to several anti-SARS-CoV-2 monoclonal antibody (mAb) therapies for the treatment of mild-to-moderate COVID-19 in patients with a high risk of progressing to severe disease. Monoclonal antibodies used to treat SARS-CoV-2 target the spike protein of the virus and block its ability to enter and infect target cells. Monoclonal antibody therapy can thus accelerate the decline in viral load and lower hospitalization rates among high-risk patients with variants susceptible to mAb therapy. However, viral resistance has been observed, in some cases leading to a transient viral rebound that can be as large as 3-4 orders of magnitude. As mAbs represent a proven treatment choice for SARS-CoV-2 and other viral infections, evaluation of treatment-emergent mAb resistance can help uncover underlying pathobiology of SARS-CoV-2 infection and may also help in the development of the next generation of mAb therapies. Although resistance can be expected, the large rebounds observed are much more difficult to explain. We hypothesize replenishment of target cells is necessary to generate the high transient viral rebound. Thus, we formulated two models with different mechanisms for target cell replenishment (homeostatic proliferation and return from an innate immune response antiviral state) and fit them to data from persons with SARS-CoV-2 treated with a mAb. We showed that both models can explain the emergence of resistant virus associated with high transient viral rebounds. We found that variations in the target cell supply rate and adaptive immunity parameters have a strong impact on the magnitude or observability of the viral rebound associated with the emergence of resistant virus. Both variations in target cell supply rate and adaptive immunity parameters may explain why only some individuals develop observable transient resistant viral rebound. Our study highlights the conditions that can lead to resistance and subsequent viral rebound in mAb treatments during acute infection.

Cryo-EM structures of the D290V mutant of the hnRNPA2 low-complexity domain suggests how D290V affects phase separation and aggregation.

Heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2) is a human ribonucleoprotein that transports RNA to designated locations for translation via its ability to phase separate. Its mutated form, D290V, is implicated in multisystem proteinopathy known to afflict two families, mainly with myopathy and Paget's disease of bone. Here, we investigate this mutant form of hnRNPA2 by determining cryo-EM structures of the recombinant D290V low complexity domain. We find that the mutant form of hnRNPA2 differs from the WT fibrils in four ways. In contrast to the WT fibrils, the PY-nuclear localization signals in the fibril cores of all three mutant polymorphs are less accessible to chaperones. Also, the mutant fibrils are more stable than WT fibrils as judged by phase separation, thermal stability, and energetic calculations. Similar to other pathogenic amyloids, the mutant fibrils are polymorphic. Thus, these structures offer evidence to explain how a D-to-V missense mutation diverts the assembly of reversible, functional amyloid-like fibrils into the assembly of pathogenic amyloid, and may shed light on analogous conversions occurring in other ribonucleoproteins that lead to neurological diseases such as amyotrophic lateral sclerosis and frontotemporal dementia.

Multicenter, Randomized Trial of a Bionic Pancreas in Type 1 Diabetes.

BACKGROUND: Currently available semiautomated insulin-delivery systems require individualized insulin regimens for the initialization of therapy and meal doses based on carbohydrate counting for routine operation. In contrast, the bionic pancreas is initialized only on the basis of body weight, makes all dose decisions and delivers insulin autonomously, and uses meal announcements without carbohydrate counting. METHODS: In this 13-week, multicenter, randomized trial, we randomly assigned in a 2:1 ratio persons at least 6 years of age with type 1 diabetes either to receive bionic pancreas treatment with insulin aspart or insulin lispro or to receive standard care (defined as any insulin-delivery method with unblinded, real-time continuous glucose monitoring). The primary outcome was the glycated hemoglobin level at 13 weeks. The key secondary outcome was the percentage of time that the glucose level as assessed by continuous glucose monitoring was below 54 mg per deciliter; the prespecified noninferiority limit for this outcome was 1 percentage point. Safety was also assessed. RESULTS: A total of 219 participants 6 to 79 years of age were assigned to the bionic-pancreas group, and 107 to the standard-care group. The glycated hemoglobin level decreased from 7.9% to 7.3% in the bionic-pancreas group and did not change (was at 7.7% at both time points) in the standard-care group (mean adjusted difference at 13 weeks, -0.5 percentage points; 95% confidence interval [CI], -0.6 to -0.3; P<0.001). The percentage of time that the glucose level as assessed by continuous glucose monitoring was below 54 mg per deciliter did not differ significantly between the two groups (13-week adjusted difference, 0.0 percentage points; 95% CI, -0.1 to 0.04; P<0.001 for noninferiority). The rate of severe hypoglycemia was 17.7 events per 100 participant-years in the bionic-pancreas group and 10.8 events per 100 participant-years in the standard-care group (P = 0.39). No episodes of diabetic ketoacidosis occurred in either group. CONCLUSIONS: In this 13-week, randomized trial involving adults and children with type 1 diabetes, use of a bionic pancreas was associated with a greater reduction than standard care in the glycated hemoglobin level. (Funded by the National Institute of Diabetes and Digestive and Kidney Diseases and others; ClinicalTrials.gov number, NCT04200313.).

Elevated numbers of infiltrating eosinophils accelerate the progression of Duchenne muscular dystrophy pathology in mdx mice.

Eosinophils, best known for their role in anti-parasitic responses, have recently been shown to actively participate in tissue homeostasis and repair. Their regulation must be tightly controlled, as their absence or hyperplasia is associated with chronic disease (e.g. asthma or inflammatory bowel disease). In the context of skeletal muscle, eosinophils play a supportive role after acute damage. Indeed, their depletion leads to strong defects in skeletal muscle regeneration and, in the absence of eosinophil-secreted interleukin (IL) 4 and IL13, fibro-adipogenic progenitors fail to support muscle stem cell proliferation. However, the role of eosinophils in muscular dystrophy remains elusive. Although it has been shown that eosinophils are present in higher numbers in muscles from mdx mice (a mouse model for Duchenne muscular dystrophy), their depletion does not affect muscle histopathology at an early age. Here, we evaluated the impact of hyper-eosinophilia on the development of fibrofatty infiltration in aged mdx mice and found that muscle eosinophilia leads to defects in muscle homeostasis, regeneration and repair, and eventually hastens death.

Stromal Rigidity Stress Accelerates Pancreatic Intraepithelial Neoplasia Progression and Chromosomal Instability via Nuclear Protein Tyrosine Kinase 2 Localization.

Because the mechanotransduction by stromal stiffness stimulates the rupture and repair of the nuclear envelope in pancreatic progenitor cells, accumulated genomic aberrations are under selection in the tumor microenvironment. Analysis of cell growth, micronuclei, and phosphorylated Ser-139 residue of the histone variant H2AX (γH2AX) foci linked to mechanotransduction pressure in vivo during serial orthotopic passages of mouse KrasLSL-G12D/+;Trp53flox/flox;Pdx1-Cre (KPC) cancer cells in the tumor and in migrating through the size-restricted 3-µm micropores. To search for pancreatic cancer cell-of-origin, analysis of single-cell data sets revealed that the extracellular matrix shaped an alternate route of acinar-ductal transdifferentiation of acinar cells into topoisomerase II α (TOP2A)-overexpressing cancer cells and derived subclusters with copy number amplifications in MYC-PTK2 (protein tyrosine kinase 2) locus and PIK3CA. High-PTK2 expression is associated with 171 differentially methylated CpG loci, 319 differentially expressed genes, and poor overall survival in The Cancer Genome Atlas-Pancreatic Adenocarcinoma cohort. Abolished RGD-integrin signaling by disintegrin KG blocked the PTK2 phosphorylation, increased cancer apoptosis, decreased vav guanine nucleotide exchange factor 1 (VAV1) expression, and prolonged overall survival in the KPC mice. Reduction of α-smooth muscle actin deposition in the CD248 knockout KPC mice remodeled the tissue stroma and down-regulated TOP2A expression in the epithelium. In summary, stromal stiffness induced the onset of cancer cells-of-origin by ectopic TOP2A expression, and the genomic amplification of MYC-PTK2 locus via alternative transdifferentiation of pancreatic progenitor cells is the vulnerability useful for disintegrin KG treatment.

Microbial intestinal dysbiosis drives long-term allergic susceptibility by sculpting an ILC2-B1 cell-innate IgE axis.

BACKGROUND: The abundance and diversity of intestinal commensal bacteria influence systemic immunity with impact on disease susceptibility and severity. For example, loss of short chain fatty acid (SCFA)-fermenting bacteria in early life (humans and mice) is associated with enhanced type 2 immune responses in peripheral tissues including the lung. OBJECTIVE: Our goal was to reveal the microbiome-dependent cellular and molecular mechanisms driving enhanced susceptibility to type 2 allergic lung disease. METHODS: We used low-dose vancomycin to selectively deplete SCFA-fermenting bacteria in wild type mice (Vanc-dys mice). We then examined the frequency and activation status of innate and adaptive immune cell lineages with and without SCFA supplementation. Finally, we used ILC2-deficient and signal transducer and activator of transcription 6 (STAT6)-deficient transgenic mouse strains to delineate the cellular and cytokine pathways leading to enhanced allergic disease susceptibility. RESULTS: Vanc-dys mice exhibit a 2-fold increase in lung ILC2 primed to produce elevated levels of interleukin (IL)-2, -5 and -13. In addition, upon IL-33 treatment, Vanc-dys lung ILC2 display a novel ability to produce high levels of IL-4. These expanded and primed ILC2 drive B1 cell expansion and IL-4-dependent production of IgE that, in turn, leads to exacerbated allergic inflammation. Importantly, these enhanced lung inflammatory phenotypes in Vanc-dys mice were reversed by administration of dietary SCFA (specifically butyrate). CONCLUSION: SCFA regulate an ILC2-B1cell-IgE axis. Early-life administration of vancomycin, an antibiotic known to deplete SCFA-fermenting gut bacteria, primes and amplifies this axis and leads to a lifelong enhanced susceptibility to type 2 allergic lung disease.

SARS-CoV-2 monoclonal antibody treatment followed by vaccination shifts human memory B cell epitope recognition suggesting antibody feedback.

Therapeutic monoclonal antibodies (mAbs) have been studied in humans, but the impact on immune memory of mAb treatment during an ongoing infection has remained unclear. We evaluated the effect of infusion of the anti-SARS-CoV-2 spike receptor binding domain (RBD) mAb bamlanivimab on memory B cells (MBCs) in SARS-CoV-2-infected individuals. Bamlanivimab treatment skewed the repertoire of memory B cells targeting Spike towards non-RBD epitopes. Furthermore, the relative affinity of RBD memory B cells was weaker in mAb-treated individuals compared to placebo-treated individuals over time. Subsequently, after mRNA COVID-19 vaccination, memory B cell differences persisted and mapped to a specific reduction in recognition of the class II RBD site, the same RBD epitope recognized by bamlanivimab. These findings indicate a substantial role of antibody feedback in regulating memory B cell responses to infection, and single mAb administration can continue to impact memory B cell responses to additional antigen exposures months later.

Safety and Efficacy of SAB-185 for Non-hospitalized Adults with COVID-19: A Randomized Clinical Trial.

BACKGROUND: To address the need for novel COVID-19 therapies, we evaluated the fully-human polyclonal antibody product SAB-185 in a phase 3 clinical trial. METHODS: Non-hospitalized high-risk adults within 7 days of COVID-19 symptom onset were randomized 1:1 to open-label SAB-185 3,840 units/kg or casirivimab/imdevimab 1200 mg. Non-inferiority comparison was undertaken for the pre-Omicron population (casirivimab/imdevimab expected to be fully active) and superiority comparison for the Omicron population (casirivimab/imdevimab not expected to be active). Primary outcomes were the composite of all-cause hospitalizations/deaths and grade ≥3 treatment-emergent adverse events (TEAEs) through day 28. Secondary outcomes included time to sustained symptom improvement and resolution. RESULTS: Enrollment was terminated early due to low hospitalization/death rates upon Omicron emergence. 733 adults were randomized, 255 included in pre-Omicron and 392 in Omicron analysis populations. Hospitalizations/deaths occurred in 6 (5.0%) and 3 (2.2%) of pre-Omicron SAB-185 and casirivimab/imdevimab arms, respectively (absolute difference [95% CI] 2.7% [-2.3%, 8.6%]), inconclusive for non-inferiority; and 5 (2.5%) versus 3 (1.5%) (absolute difference 1.0% [-2.3%, 4.5%]) for Omicron. Risk ratios for grade ≥3 TEAEs were 0.94 [0.52, 1.71] (pre-Omicron) and 1.71 [0.96, 3.07] (Omicron). Time to symptom improvement and resolution were shorter for SAB-185, median 11 vs 14 (pre-Omicron) and 11 vs 13 days (Omicron) (symptom improvement), and 16 vs 24 days and 18 vs >25 days (symptom resolution), p<0.05 for symptom resolution for Omicron only. CONCLUSIONS: SAB-185 had an acceptable safety profile with faster symptom resolution in the Omicron population. Additional studies are needed to characterize its efficacy for COVID-19.

Characterization of Treatment Resistance and Viral Kinetics in the Setting of Single-Active Versus Dual-Active Monoclonal Antibodies Against Severe Acute Respiratory Syndrome Coronavirus 2.

BACKGROUND: Monoclonal antibodies (mAbs) represent a crucial antiviral strategy for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, but it is unclear whether combination mAbs offer a benefit over single-active mAb treatment. Amubarvimab and romlusevimab significantly reduced the risk of hospitalizations or death in the ACTIV-2/A5401 trial. Certain SARS-CoV-2 variants are intrinsically resistant against romlusevimab, leading to only single-active mAb therapy with amubarvimab in these variants. We evaluated virologic outcomes in individuals treated with single- versus dual-active mAbs. METHODS: Participants were nonhospitalized adults at higher risk of clinical progression randomized to amubarvimab plus romlusevimab or placebo. Quantitative SARS-CoV-2 RNA levels and targeted S-gene next-generation sequencing was performed on anterior nasal samples. We compared viral load kinetics and resistance emergence between individuals treated with effective single- versus dual-active mAbs depending on the infecting variant. RESULTS: Study participants receiving single- or dual-active mAbs had similar demographics, baseline nasal viral load, symptom score, and symptom duration. Compared with single-active mAb treatment, treatment with dual-active mAbs led to faster viral load decline at study days 3 (P < .001) and 7 (P < .01). Treatment-emergent resistance mutations were more likely to be detected after amubarvimab plus romlusevimab treatment than with placebo (2.6% vs 0%; P < .001) and were more frequently detected in the setting of single-active compared with dual-active mAb treatment (7.3% vs 1.1%; P < .01). Single-active and dual-active mAb treatment resulted in similar decrease in rates of hospitalizations or death. CONCLUSIONS: Compared with single-active mAb therapy, dual-active mAbs led to similar clinical outcomes but significantly faster viral load decline and a lower risk of emergent resistance.

Adverse Pregnancy Outcomes Among Women with Human Immunodeficiency Virus Taking Isoniazid Preventive Therapy During the First Trimester.

BACKGROUND: Isoniazid preventive therapy (IPT) is recommended for tuberculosis prevention yet data on the safety of first-trimester pregnancy exposure are limited. METHODS: Planned secondary analysis in a TB prevention trial of adverse pregnancy outcomes among participants assigned to 9-month IPT who became pregnant during (IPT-exposed) or after (unexposed) IPT. Regression models compared binary outcomes of a composite adverse outcome (any non-live birth, excluding induced abortion); preterm delivery <37 weeks; and low birth weight <2500 g) among exposure groups. Models were adjusted for latent TB infection, maternal age, CD4 count, and antiretroviral therapy (ART). RESULTS: In total, 128 participants had a known pregnancy outcome; 39 IPT-exposed and 89 unexposed. At pregnancy outcome, ART use was lower in IPT-exposed (79%) than unexposed women (98%). Overall, 29 pregnancies ended in a composite adverse outcome (25 spontaneous abortions, 2 stillbirths and 2 ectopic pregnancies), 15 preterm deliveries, and 10 infants with low birth weight. IPT was associated with the composite adverse outcome adjusting for covariates at enrollment (adjusted relative risk [aRR] 1.98; 95% confidence interval [CI] 1.15, 3.41), but the effect was attenuated when adjusted for covariates at pregnancy outcome (aRR 1.47; 95% CI .84, 2.55); IPT was not associated with preterm delivery (relative risk [RR] 0.87; 95% CI .32-2.42) or low birth weight (RR 1.01; 95% CI .29, 3.56). CONCLUSIONS: First-trimester IPT exposure was associated with nearly two-fold increased risk of fetal demise, mostly spontaneous abortion, though the association was attenuated when adjusted for covariates proximal to pregnancy outcome including ART use. Further study is needed to inform TB prevention guidelines.

Severe Acute Respiratory Syndrome Coronavirus 2 Plasma Antibody and Nucleocapsid Antigen Status Predict Outcomes in Outpatients With Coronavirus Disease 2019.

BACKGROUND: Reliable biomarkers of coronavirus disease 2019 (COVID-19) outcomes are critically needed. We evaluated associations of spike antibody (Ab) and plasma nucleocapsid antigen (N Ag) with clinical outcomes in nonhospitalized persons with mild-to-moderate COVID-19. METHODS: Participants were nonhospitalized adults with mild-to-moderate COVID-19 enrolled in ACTIV-2 between January and July 2021 and randomized to placebo. We used quantitative assays for severe acute respiratory syndrome coronavirus 2 spike Ab and N Ag in blood and determined numbers of hospitalization/death events within 28 days and time to symptom improvement. RESULTS: Of 209 participants, 77 (37%) had quantifiable spike Ab and 139 (67%) quantifiable N Ag. Median age was 50 years; 111 (53%) were female, 182 (87%) White, and 105 (50%) Hispanic/Latino. Higher risk of hospitalization/death was seen with unquantifiable (22/132 [16.7%]) versus quantifiable (1/77 [1.3%]) spike Ab (risk ratio [RR], 12.83 [95% confidence interval {CI}, 1.76-93.34]) and quantifiable (22/139 [15.8%]) vs unquantifiable (1/70 [1.4%]) N Ag (RR, 11.08 [95% CI, 1.52-80.51]). Increasing risk of hospitalizations/deaths was seen with increasing N Ag levels. Time to symptom improvement was longer with unquantifiable versus quantifiable spike Ab (median, 14 [interquartile range {IQR}, 8 to >27] vs 8 [IQR, 4-22] days; adjusted hazard ratio [aHR], 0.66 [95% CI, .45-.96]) and with quantifiable versus unquantifiable N Ag (median, 12 [7 to >27] vs 10 [5-22] days; aHR, 0.79 [95% CI, .52-1.21]). CONCLUSIONS: Absence of spike Ab and presence of plasma N Ag predicted hospitalization/death and delayed symptom improvement in COVID-19 outpatients.

Effectiveness of double-dose dolutegravir in people receiving rifampin-based tuberculosis treatment: an observational, cohort study of people with HIV from six countries.

BACKGROUND: Tenofovir-lamivudine-dolutegravir (TLD) is the preferred first-line antiretroviral therapy (ART) regimen. An additional 50 mg dose of dolutegravir (TLD + 50) is required with rifampin-containing tuberculosis (TB) co-treatment. There are limited data on the effectiveness of TLD + 50 in individuals with TB/HIV. METHODS: Prospective, observational cohort study at 12 sites in Haiti, Kenya, Malawi, South Africa, Uganda, Zimbabwe. Participants starting TLD and rifampin-containing TB treatment were eligible. Primary outcome was HIV-1 RNA ≤1000 copies/mL at end of TB treatment. FINDINGS: We enrolled 91 participants with TB/HIV: 75 (82%) ART-naïve participants starting TLD after a median 15 days on TB treatment, 10 (11%) ART-naïve participants starting TLD and TB treatment, 5 (5%) starting TB treatment after a median 3.3 years on TLD, and 1 (1%) starting TB treatment and TLD after changing from efavirenz/lamivudine/tenofovir. Median age was 37 years, 35% female, median CD4 count 120 cells/mm3 (IQR 50-295), 87% had HIV-1 RNA >1000 copies/mL. Two participants died during TB treatment. Among 89 surviving participants, 80 were followed to TB treatment completion, including 7 who had no HIV-1 RNA result due to missed visits. Primary virologic outcome was assessed in 73 participants, of whom 69 (95%, 95% CI 89-100%) had HIV-1 RNA ≤1000 copies/mL. No dolutegravir resistance mutations were detected among four participants with HIV-1 RNA >1000 copies/mL. INTERPRETATION: In routine programmatic settings, concurrent rifampin-containing TB treatment and TLD + 50 was feasible, well-tolerated, and achieved high rates of viral suppression in a cohort of predominantly ART-naïve people with TB/HIV.

Development of a measuring app for systemic sclerosis-related digital ulceration (SALVE: Scleroderma App for Lesion VErification).

OBJECTIVES: To test the hypothesis that photographs (in addition to self-reported data) can be collected daily by patients with systemic sclerosis (SSc) using a smartphone app designed specifically for digital lesions, and could provide an objective outcome measure for use in clinical trials. METHODS: An app was developed to collect images and patient reported outcome measures (PROMS) including Pain score and the Hand Disability in Systemic Sclerosis-Digital Ulcers (HDISS-DU) questionnaire. Participants photographed their lesion(s) each day for 30 days and uploaded images to a secure repository. Lesions were analysed both manually and automatically, using a machine learning approach. RESULTS: 25 patients with SSc-related digital lesions consented of whom 19 completed the 30-day study, with evaluable data from 27 lesions. Mean (standard deviation [SD]) baseline Pain score was 5.7 (2.4) and HDISS-DU 2.2 (0.9), indicating high lesion and disease-related morbidity. 506 images were used in the analysis (mean number of used images per lesion 18.7, SD 8.3). Mean (SD) manual and automated lesion areas at day 1 were 11.6 (16.0) and 13.9 (16.7) mm2 respectively. Manual area decreased by 0.08mm2 per day (2.4mm2 over 30 days) and automated area by 0.1mm2 (3.0mm2 over 30 days). Average gradients of manual and automated measurements over 30 days correlated strongly (r = 0.81). Manual measurements were on average 40% lower than automated, with wide limits of agreement. CONCLUSION: Even patients with significant hand disability were able to use the app. Automated measurement of finger lesions could be valuable as an outcome measure in clinical trials.

Patients' unmet needs and treatment preferences concerning digital ulcers in systemic sclerosis.

INTRODUCTION: Digital ulcers (DUs) significantly impact on quality of life and function in patients with systemic sclerosis (SSc). The aim of our survey was to explore patients' perspectives and their unmet needs concerning SSc-DUs. MATERIALS: SSc patients were invited through international patient associations and social media to participate in an online survey. RESULTS: 358 responses were obtained from 34 countries: US (65.6%), UK (11.5%) and Canada (4.5%). Recurrent DUs are common: >10 DUs (46.1%), 5-10 DUs (21.5%), 1-5 DUs (28.5%), 1 DU (3.9%). Fingertip DUs were most frequent (84.9%), followed by those overlying the interphalangeal joints (50.8%). The impact of DUs in patients is broad, from broad-ranging emotional impacts to impact on activities of daily living, and personal relationships. Half (51.7%) of respondents reported that they received wound/ulcer care, most often provided by non-specialist wound care clinics (63.8%). There was significant variation in local (wound) DU care, in particular the use of debridement and pain management. DU-related education was only provided to one-third of patients. One-quarter (24.6%) were 'very satisfied' or 'satisfied' that the provided DU treatment(s) relieved their DU symptoms. Pain, limited hand function, and ulcer duration/chronicity were the main reasons for patients to consider changing DU treatment. CONCLUSIONS: Our data show that there is a large variation in DU treatment between countries. Patient access to specialist wound-care services is limited and only a small proportion of patients had their DU needs met. Moreover, patient education is often neglected. Evidence-based treatment pathways are urgently needed for DU management.

Dielectric properties of human macrophages are altered by Mycobacterium tuberculosis infection.

The analysis of cell electrophysiology for pathogenic samples at BSL3 can be problematic. It is virtually impossible to isolate infected from uninfected without a label, for example green fluorescent protein, which can potentially alter the cell electrical properties. Furthermore, the measurement of highly pathogenic organisms often requires equipment dedicated only for use with these organisms due to safety considerations. To address this, we have used dielectrophoresis to study the electrical properties of the human THP-1 cell line and monocyte-derived macrophages before and after infection with non-labelled Mycobacterium tuberculosis. Infection with these highly pathogenic bacilli resulted in changes including a raised surface conductance (associated with reduced zeta potential) and increased capacitance, suggesting an increase in surface roughness. We have also investigated the effect of fixation on THP-1 cells as a means to enable study on fixed samples in BSL1 or 2 laboratories, which suggests that the properties of these cells are largely unaffected by the fixation process. This advance results in a novel technique enabling the isolation of infected and non-infected cells in a sample without labelling.

On the low-frequency dispersion observed in dielectrophoresis spectra.

The foundation of dielectrophoresis (DEP) as a tool for biological investigation is the use of the Clausius-Mossotti (C-M) factor to model the observed behaviour of cells experiencing DEP across a frequency range. Nevertheless, it is also the case that at lower frequencies, the DEP spectrum deviates from predictions; there exists a rise in DEP polarisability, which varies in frequency and magnitude with different cell types and medium conductivities. In order to evaluate the origin of this effect, we have studied DEP spectra from five cell types (erythrocytes, platelets, neurons, HeLa cancer cells and monocytes) in several conditions including medium conductivity and cell treatment. Our results suggest the effect manifests as a low-pass dispersion whose cut-off frequency varies with membrane conductance and capacitance as determined using the DEP spectrum; the effect also varies as a logarithm of medium conductivity and Debye length. These together suggest that the values of membrane capacitance and conductance depend not only on the impedance of the membrane itself, but also of the surrounding double layer. The amplitude of the effect in different cell types compared to the C-M factor was found to correlate with the depolarisation factors for the cells' shapes, suggesting that this ratio may be useful as an indicator of cell shape for DEP modelling.

Embedding a novel screening programme for sexually transmitted infections (chlamydia and gonorrhoea) within an ambulatory emergency surgical assessment unit: an observational cohort study.

BACKGROUND: A number of females with pelvic inflammatory disease will present to general surgical services with non-specific abdominal pain. Screening for sexually transmitted infections (STI) as an underlying cause is not routinely offered. We therefore established an STI screening programme for young females presenting to a same day emergency ambulatory surgical clinic as part of the diagnostic pathway. Data outlining the incidence and prevalence of STIs as the underlying cause of lower abdominal pain were collected. METHODS: We conducted an observational cohort study. Self-collected vulvovaginal swabs for chlamydia and gonorrhoea were offered as part of a standardised diagnostic pathway for all females meeting inclusion criteria presenting with abdominal pain. Positive results were referred to our local sexual health team for treatment and contact tracing. RESULTS: The cohort comprised 297 eligible patients; 259 participated, 20 patients declined testing and 18 samples were rejected as inadequate in the laboratory. 5.4% of swab results were positive (2 gonorrhoea and 12 chlamydia). All patients with positive swabs had presented with lower abdominal pain and of these only 21% had a documented sexual history. CONCLUSION: Undiagnosed STIs are prevalent, with significant fertility and public health risks. Young females seeking medical assessment for abdominal pain provide an opportunistic screening cohort with a likely subset of patients presenting with abdominal pain as a direct result of an STI. Our results demonstrate a high incidence of positive tests, suggesting further training of surgeons to include a sexual history in assessment of females with abdominal pain is vital.

Biomechanics of circulating cellular and subcellular bioparticles: beyond separation.

Biomechanical attributes have emerged as novel markers, providing a reliable means to characterize cellular and subcellular fractions. Numerous studies have identified correlations between these factors and patients' medical status. However, the absence of a thorough overview impedes their applicability in contemporary state-of-the-art therapeutic strategies. In this context, we provide a comprehensive analysis of the dimensions, configuration, rigidity, density, and electrical characteristics of normal and abnormal circulating cells. Subsequently, the discussion broadens to encompass subcellular bioparticles, such as extracellular vesicles (EVs) enriched either from blood cells or other tissues. Notably, cell sizes vary significantly, from 2 µm for platelets to 25 µm for circulating tumor cells (CTCs), enabling the development of size-based separation techniques, such as microfiltration, for specific diagnostic and therapeutic applications. Although cellular density is relatively constant among different circulating bioparticles, it allows for reliable density gradient centrifugation to isolate cells without altering their native state. Additionally, variations in EV surface charges (-6.3 to -45 mV) offer opportunities for electrophoretic and electrostatic separation methods. The distinctive mechanical properties of abnormal cells, compared to their normal counterparts, present an exceptional opportunity for diverse medical and biotechnological approaches. This review also aims to provide a holistic view of the current understanding of popular techniques in this domain that transcend conventional boundaries, focusing on early harvesting of malignant cells from body fluids, designing effective therapeutic options, cell targeting, and resonating with tissue and genetic engineering principles.