PI 3-Kinase and the Histone Methyl-Transferase KMT2D Collaborate to Induce Arp2/3-Dependent Migration of Mammary Epithelial Cells.

Breast cancer develops upon sequential acquisition of driver mutations in mammary epithelial cells; however, how these mutations collaborate to transform normal cells remains unclear in most cases. We aimed to reconstitute this process in a particular case. To this end, we combined the activated form of the PI 3-kinase harboring the H1047R mutation with the inactivation of the histone lysine methyl-transferase KMT2D in the non-tumorigenic human mammary epithelial cell line MCF10A. We found that PI 3-kinase activation promoted cell-cycle progression, especially when growth signals were limiting, as well as cell migration, both in a collective monolayer and as single cells. Furthermore, we showed that KMT2D inactivation had relatively little influence on these processes, except for single-cell migration, which KMT2D inactivation promoted in synergy with PI 3-kinase activation. The combination of these two genetic alterations induced expression of the ARPC5L gene that encodes a subunit of the Arp2/3 complex. ARPC5L depletion fully abolished the enhanced migration persistence exhibited by double-mutant cells. Our reconstitution approach in MCF10A has thus revealed both the cell function and the single-cell migration, and the underlying Arp2/3-dependent mechanism, which are synergistically regulated when KMT2D inactivation is combined with the activation of the PI 3-kinase.

Investigating surface proteins and antibody combinations for detecting circulating tumor cells of various sarcomas.

Circulating tumor cells (CTCs) have gathered attention as a biomarker for carcinomas. However, CTCs in sarcomas have received little attention. In this work, we investigated cell surface proteins and antibody combinations for immunofluorescence detection of sarcoma CTCs. A microfluidic device that combines filtration and immunoaffinity using gangliosides 2 and cell surface vimentin (CSV) antibodies was employed to capture CTCs. For CTC detection, antibodies against cytokeratins 7 and 8 (CK), pan-cytokeratin (panCK), or a combination of panCK and CSV were used. Thirty-nine blood samples were collected from 21 patients of various sarcoma subtypes. In the independent samples study, samples were subjected to one of three antibody combination choices. Significant difference in CTC enumeration was found between CK and panCK + CSV, and between panCK and panCK + CSV. Upon stratification of CK+ samples, those of metastatic disease had a higher CTC number than those of localized disease. In the paired samples study involving cytokeratin-positive sarcoma subtypes, using panCK antibody detected more CTCs than CK. Similarly, for osteosarcoma, using panCK + CSV combination resulted in a higher CTC count than panCK. This study emphasized deliberate selection of cell surface proteins for sarcoma CTC detection and subtype stratification for studying cancers as heterogeneous as sarcomas.

Observation of Seven Astrophysical Tau Neutrino Candidates with IceCube.

We report on a measurement of astrophysical tau neutrinos with 9.7 yr of IceCube data. Using convolutional neural networks trained on images derived from simulated events, seven candidate ν_{τ} events were found with visible energies ranging from roughly 20 TeV to 1 PeV and a median expected parent ν_{τ} energy of about 200 TeV. Considering backgrounds from astrophysical and atmospheric neutrinos, and muons from π^{±}/K^{±} decays in atmospheric air showers, we obtain a total estimated background of about 0.5 events, dominated by non-ν_{τ} astrophysical neutrinos. Thus, we rule out the absence of astrophysical ν_{τ} at the 5σ level. The measured astrophysical ν_{τ} flux is consistent with expectations based on previously published IceCube astrophysical neutrino flux measurements and neutrino oscillations.

Features of trastuzumab-related cardiac dysfunction: deformation analysis outside left ventricular global longitudinal strain.

Background: Cancer therapy-related cardiac dysfunction due to trastuzumab has been well-known for many years, and echocardiographic surveillance is recommended every 3 months in patients undergoing trastuzumab treatment, irrespective of the baseline cardiotoxicity risk. However, the potential harm and cost of overscreening in low- and moderate-risk patients have become great concerns. Objectives: This study aimed to identify the incidence of early cancer therapy-related cardiac dysfunction (CTRCD) and the behaviours of left and right heart deformations during trastuzumab chemotherapy in low- and moderate-risk patients. Methods: We prospectively enrolled 110 anthracycline-naïve women with breast cancer and cardiovascular risk factors who were scheduled to receive trastuzumab. The left ventricular ejection fraction (LVEF), left ventricular global longitudinal strain (LV-GLS), and right ventricular and left atrial longitudinal strains were evaluated using echocardiography at baseline, before every subsequent cycle and 3 weeks after the final dose of trastuzumab. The baseline risk of CTRCD was graded according to the risk score proposed by the Heart Failure Association (HFA) Cardio-Oncology Working Group and the International Cardio-Oncology Society (ICOS). CTRCD and its severity were defined according to the current European Society of Cardiology (ESC) guidelines. Results: Twelve (10.9%) patients had asymptomatic CTRCD. All CTRCD occurred sporadically during the first 9 months of the active trastuzumab regimen in both low- and moderate-risk patients. While CTRCD was graded as moderate severity in 41.7% of patients and heart failure therapy was initiated promptly, no irreversible cardiotoxicity or trastuzumab interruption was recorded at the end of follow-up. Among the left and right heart deformation indices, only LV-GLS decreased significantly in the CTRCD group during the trastuzumab regimen. Conclusions: CTRCD is prevalent in patients with non-high-risk breast cancer undergoing trastuzumab chemotherapy. Low- and moderate-risk patients show distinct responses to trastuzumab. The LV-GLS is the only deformation index sensitive to early trastuzumab-related cardiac dysfunction.

Exploring COVID-19 education to support vaccine confidence amongst the general adult population with special considerations for healthcare and long-term care staff: A scoping review.

Background: Despite the demonstrated efficacy of approved COVID-19 vaccines, high levels of hesitancy were observed in the first few months of the COVID-19 vaccines' rollout. Factors contributing to vaccine hesitancy are well-described in the literature. Among the various strategies for promoting vaccine confidence, educational interventions provide a foundationally and widely implemented set of approaches for supporting individuals in their vaccine decisions. However, the evidence around the measurable impact of various educational strategies to improve vaccine confidence is limited. We conducted a scoping review with the aim of exploring and characterizing educational interventions delivered during the pandemic to support COVID-19 vaccine confidence in adults. Methods: We developed a search strategy with a medical information scientist and searched five databases, including Ovid MEDLINE and Web of Science, as well as grey literature. We considered all study designs and reports. Interventions delivered to children or adolescents, interventions on non-COVID-19 vaccines, as well as national or mass vaccination campaigns without documented interaction(s) between facilitator(s) and a specific audience were excluded. Articles were independently screened by three reviewers. After screening 4602 titles and abstracts and 174 full-text articles across two rounds of searches, 22 articles met our inclusion criteria. Ten additional studies were identified through hand searching. Data from included studies were charted and results were described narratively. Results: We included 32 studies and synthesized their educational delivery structure, participants (i.e., facilitators and priority audience), and content. Formal, group-based presentations were the most common type of educational intervention in the included studies (75%). A third of studies (34%) used multiple strategies, with many formal group-based presentations being coupled with additional individual-based interventions (29%). Given the novelty of the COVID-19 vaccines and the unique current context, studies reported personalized conversations, question periods, and addressing misinformation as important components of the educational approaches reviewed. Conclusions: Various educational interventions were delivered during the COVID-19 pandemic, with many initiatives involving multifaceted interventions utilizing both formal and informal approaches that leveraged community (cultural, religious) partnerships when developing and facilitating COVID-19 vaccine education. Train-the-trainer approaches with recognized community members could be of value as trust and personal connections were identified as strong enablers throughout the review.

Microfluidics-Enabled Isolation and Single-Cell Analysis of Circulating Tumor Cells.

Microfluidic platforms enable the enrichment and analysis of circulating tumor cells (CTCs), a potential biomarker for cancer diagnosis, prognosis, and theragnosis. Combined with immunocytochemistry/immunofluorescence (ICC/IF) assays for CTCs, microfluidics-enabled detection presents a unique opportunity to study tumor heterogeneity and predict treatment response, both of which can help cancer drug development. In this chapter, we detail the protocols and methods employed to fabricate and use a microfluidic device for the enrichment, detection, and analysis of single CTCs from the blood samples of sarcoma patients.

Role of Electrical Delay in Cardiac Resynchronization Therapy Response.

Traditionally, left ventricular (LV) lead position was guided by anatomic criteria of pacing from the lateral wall of the LV. However, large trials showed little effect of LV lead position on outcomes, other than noting worse outcomes with apical positions. Given the poor correlation of cardiac resynchronization therapy (CRT) outcomes with anatomically guided LV lead placement, focus shifted toward more physiologic predictors such as targeting the areas of delayed mechanical and electrical activation. Measures of left ventricular delay and interventricular delay are strong predictors of CRT response.

Longitudinal Study of Circulating Biomarkers in Patients with Resectable Pancreatic Ductal Adenocarcinoma.

While patients with resectable pancreatic ductal adenocarcinoma (PDAC) show improved survival compared to their non-resectable counterparts, survival remains low owing to occult metastatic disease and treatment resistance. Liquid biopsy based on circulating tumor cells (CTCs) and cell-free DNA (cfDNA) has been shown to predict recurrence and treatment resistance in various types of cancers, but their utility has not been fully demonstrated in resectable PDAC. We have simultaneously tracked three circulating biomarkers, including CTCs, cfDNA, and circulating tumor DNA (ctDNA), over a period of cancer treatment using a microfluidic device and droplet digital PCR (ddPCR). The microfluidic device is based on the combination of filtration and immunoaffinity mechanisms. We have measured CTCs, cfDNA, and ctDNA in a cohort of seven resectable PDAC patients undergoing neoadjuvant therapy followed by surgery, and each patient was followed up to 10 time points over a period of 4 months. CTCs were detectable in all patients (100%) at some point during treatment but were detectable in only three out of six patients (50%) prior to the start of treatment. Median cfDNA concentrations remained comparable to negative controls throughout treatment. ddPCR was able to find KRAS mutations in six of seven patients (86%); however, these mutations were present in only two of seven patients (29%) prior to treatment. Overall, the majority of circulating biomarkers (81% for CTCs and 91% for cfDNA/ctDNA) were detected after the start of neoadjuvant therapy but before surgery. This study suggests that a longitudinal study of circulating biomarkers throughout treatment provides more useful information than those single time-point tests for resectable PDAC patients.

Development and Evaluation of a Molecular Hepatitis A Virus Assay for Serum and Stool Specimens.

Hepatitis A virus (HAV) is an emerging public health concern and there is an urgent need for ways to rapidly identify cases so that outbreaks can be managed effectively. Conventional testing for HAV relies on anti-HAV IgM seropositivity. However, studies estimate that 10-30% of patients may not be diagnosed by serology. Molecular assays that can directly detect viral nucleic acids have the potential to improve diagnosis, which is key to prevent the spread of infections. In this study, we developed a real-time PCR (RT-PCR) assay to detect HAV RNA for the identification of acute HAV infection. Primers were designed to target the conserved 5'-untranslated region (5'-UTR) of HAV, and the assay was optimized on both the Qiagen Rotor-Gene and the BD MAX. We successfully detected HAV from patient serum and stool samples with moderate differences in sensitivity and specificity depending on the platform used. Our results highlight the clinical utility of using a molecular assay to detect HAV from various specimen types that can be implemented in hospitals to assist with diagnostics, treatment and prevention.

Streptococcus pyogenes secondary impetigo due to loofah sponge use.

Loofah sponges have been implicated in skin and soft tissue infections due to their ability to harbor bacteria and cause microtrauma to the skin. In this case report, we describe a case of impetigo and cellulitis due to Streptococcus pyogenes complicated by secondary spread through loofah sponge use. The same organism was cultured from the infected body sites and loofah sponge, and a comparative genomic analysis confirmed that the isolates were identical.

Emerging and divergent roles of pyrophosphorylated nucleotides in bacterial physiology and pathogenesis.

Bacteria inhabit diverse environmental niches and consequently must modulate their metabolism to adapt to stress. The nucleotide second messengers guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) (collectively referred to as (p)ppGpp) are essential for survival during nutrient starvation. (p)ppGpp is synthesized by the RelA-SpoT homologue (RSH) protein family and coordinates the control of cellular metabolism through its combined effect on over 50 proteins. While the role of (p)ppGpp has largely been associated with nutrient limitation, recent studies have shown that (p)ppGpp and related nucleotides have a previously underappreciated effect on different aspects of bacterial physiology, such as maintaining cellular homeostasis and regulating bacterial interactions with a host, other bacteria, or phages. (p)ppGpp produced by pathogenic bacteria facilitates the evasion of host defenses such as reactive nitrogen intermediates, acidic pH, and the complement system. Additionally, (p)ppGpp and pyrophosphorylated derivatives of canonical adenosine nucleotides called (p)ppApp are emerging as effectors of bacterial toxin proteins. Here, we review the RSH protein family with a focus on its unconventional roles during host infection and bacterial competition.

Evaluation of the effect of 3D porous Chitosan-alginate scaffold stiffness on breast cancer proliferation and migration.

Breast cancer (BCa) is one of the most common cancers for women and metastatic BCa causes the majority of deaths. The extracellular matrix (ECM) stiffens during cancer progression and provides biophysical signals to modulate proliferation, morphology, and metastasis. Cells utilize mechanotransduction and integrins to sense and respond to ECM stiffness. Chitosan-alginate (CA) scaffolds have been used for 3D culture, but lack integrin binding ligands, resulting in round cell morphology and limited cell-material interaction. In this study, 2, 4, and 6 wt% CA scaffolds were produced to mimic the stages of BCa progression and evaluate the BCa response to CA scaffold stiffness. All three CA scaffold compositions highly porous with interconnected pores and scaffold stiffness increased with increasing polymer concentration. MDA-MB-231 (231) cells were cultured in CA scaffolds and 2D cultures for 7 d. All CA scaffold cultures had similar cell numbers at 7 d and the 231 cells formed clusters that increased in size during the culture. The 2 wt% CA had the largest clusters throughout the 7 d culture compared with the 4 and 6 wt% CA. The 231 cell migration was evaluated on 2D surfaces after 7 d culture. The 6 wt% CA cultured cells had the greatest migration speed, followed by 4 wt% CA, 2D cultures, and 2 wt% CA. These results suggest that 231 cells sensed the stiffness of CA scaffolds without the presence of focal adhesions. This indicates that a non-integrin-based mechanism may explain the observed mechanotransduction response.

Exosome isolation using nanostructures and microfluidic devices.

Exosomes contain cargoes of proteins, lipids, micro-ribonucleic acids, and functional messenger RNAs, and they play a key role in cell-to-cell communication and hold valuable information about biological processes such as disease pathology. To harvest their potentials in disease diagnostics, prognostics, and therapeutics, exosome isolation is a crucial first step in providing pure and intact samples for both research and clinical purposes. Unfortunately, conventional methods for exosome separation suffer from low purity, low capture efficiency, long processing time, large sample volume requirement, the need for dedicated equipment and trained personnel, and high cost. In the last decade, microfluidic devices, especially those that incorporate nanostructures, have emerged as superior alternatives for exosome isolation and detection. In this review, we examine microfluidic platforms, dividing them into six categories based on their capture mechanisms: passive-structure-based affinity, immunomagnetic-based affinity, filtration, acoustofluidics, electrokinetics, and optofluidics. Here, we start out exploring the research and clinical needs that translate into important performance parameters for new exosome isolation designs. Then, we briefly introduce the conventional methods and discuss how their failure to meet those performance standards sparks an intense interest in microfluidic device innovations. The essence of this review is to lead an in-depth discussion on not only the technicality of those microfluidic platforms, but also their strengths and weaknesses with regards to the performance parameters set forth. To close the conversation, we call for the inclusion of exosome confirmation and contamination evaluation as part of future device development and performance assessment process, so that collectively, efforts towards microfluidics and nanotechnology for exosome isolation and analysis may soon see the light of real-world applications.

Environmental factors influence the local establishment of Wolbachia in Aedes aegypti mosquitoes in two small communities in central Vietnam.

Background: The wMel strain of Wolbachia has been successfully introduced into Aedes aegypti mosquitoes and subsequently shown to reduce transmission of dengue and other pathogens, under both laboratory and field conditions. Here we describe the entomological outcomes of wMel Wolbachia mosquito releases in two small communities in Nha Trang City in central Vietnam. Methods: The wMel strain of Wolbachia was backcrossed into local Aedes aegypti genotype and mosquito releases were undertaken by community members or by staff. Field monitoring was undertaken to track Wolbachia establishment in local Ae. aegypti mosquito populations. Ecological studies were undertaken to assess relationships between environmental factors and the spatial and temporal variability in Wolbachia infection prevalence in mosquitoes. Results: Releases of wMel Wolbachia Ae. aegypti mosquitoes in two small communities in Nha Trang City resulted in the initial establishment of Wolbachia in the local Ae. aegypti mosquito populations, followed by seasonal fluctuations in Wolbachia prevalence. There was significant small-scale spatial heterogeneity in Wolbachia infection prevalence in the Tri Nguyen Village site, resulting in the loss of wMel Wolbachia infection in mosquitoes in north and center areas, despite Wolbachia prevalence remaining high in mosquitoes in the south area. In the second site, Vinh Luong Ward, Wolbachia has persisted at a high level in mosquitoes throughout this site despite similar seasonal fluctuations in wMel Wolbachia prevalence. Conclusion: Seasonal variation in Wolbachia infection prevalence in mosquitoes was associated with elevated temperature conditions, and was possibly due to imperfect maternal transmission of Wolbachia. Heterogeneity in Wolbachia infection prevalence was found throughout one site, and indicates additional factors may influence Wolbachia establishment.

(p)ppGpp-Dependent Regulation of the Nucleotide Hydrolase PpnN Confers Complement Resistance in Salmonella enterica Serovar Typhimurium.

The stringent response is an essential mechanism of metabolic reprogramming during environmental stress that is mediated by the nucleotide alarmones guanosine tetraphosphate and pentaphosphate [(p)ppGpp]. In addition to physiological adaptations, (p)ppGpp also regulates virulence programs in pathogenic bacteria, including Salmonella enterica serovar Typhimurium. S Typhimurium is a common cause of acute gastroenteritis, but it may also spread to systemic tissues, resulting in severe clinical outcomes. During infection, S Typhimurium encounters a broad repertoire of immune defenses that it must evade for successful host infection. Here, we examined the role of the stringent response in S Typhimurium resistance to complement-mediated killing and found that the (p)ppGpp synthetase-hydrolase, SpoT, is required for bacterial survival in human serum. We identified the nucleotide hydrolase, PpnN, as a target of the stringent response that is required to promote bacterial fitness in serum. Using chromatography and mass spectrometry, we show that PpnN hydrolyzes purine and pyrimidine monophosphates to generate free nucleobases and ribose 5'-phosphate, and that this metabolic activity is required for conferring resistance to complement killing. In addition to PpnN, we show that (p)ppGpp is required for the biosynthesis of the very long and long O-antigen in the outer membrane, known to be important for complement resistance. Our results provide new insights into the role of the stringent response in mediating evasion of the innate immune system by pathogenic bacteria.

Longitudinal sampling is required to maximize detection of intrahost A/H3N2 virus variants.

Seasonal human influenza viruses continually change antigenically to escape from neutralizing antibodies. It remains unclear how genetic variation in the intrahost virus population and selection at the level of individual hosts translates to the fast-paced evolution observed at the global level because emerging intrahost antigenic variants are rarely detected. We tracked intrahost variants in the hemagglutinin and neuraminidase surface proteins using longitudinally collected samples from 52 patients infected by A/H3N2 influenza virus, mostly young children, who received oseltamivir treatment. We identified emerging putative antigenic variants and oseltamivir-resistant variants, most of which remained detectable in samples collected at subsequent days, and identified variants that emerged intrahost immediately prior to increases in global rates. In contrast to most putative antigenic variants, oseltamivir-resistant variants rapidly increased to high frequencies in the virus population. Importantly, the majority of putative antigenic variants and oseltamivir-resistant variants were first detectable four or more days after onset of symptoms or start of treatment, respectively. Our observations demonstrate that de novo variants emerge, and may be positively selected, during the course of infection. Additionally, based on the 4-7 days post-treatment delay in emergence of oseltamivir-resistant variants in six out of the eight individuals with such variants, we find that limiting sample collection for routine surveillance and diagnostic testing to early timepoints after onset of symptoms can potentially preclude detection of emerging, positively selected variants.

Value of lipocalin 2 as a potential biomarker for bacterial meningitis.

OBJECTIVES: Central nervous system (CNS) infections are common causes of morbidity and mortality worldwide. We aimed to discover protein biomarkers that could rapidly and accurately identify the likely cause of the infections, essential for clinical management and improving outcome. METHODS: We applied liquid chromatography tandem mass spectrometry on 45 cerebrospinal fluid (CSF) samples from a cohort of adults with and without CNS infections to discover potential diagnostic biomarkers. We then validated the diagnostic performance of a selected biomarker candidate in an independent cohort of 364 consecutively treated adults with CNS infections admitted to a referral hospital in Vietnam. RESULTS: In the discovery cohort, we identified lipocalin 2 (LCN2) as a potential biomarker of bacterial meningitis (BM) other than tuberculous meningitis. The analysis of the validation cohort showed that LCN2 could discriminate BM from other CNS infections (including tuberculous meningitis, cryptococcal meningitis and virus/antibody-mediated encephalitis), with sensitivity of 0.88 (95% confident interval (CI), 0.77-0.94), specificity of 0.91 (95% CI, 0.88-0.94) and diagnostic odds ratio of 73.8 (95% CI, 31.8-171.4). LCN2 outperformed other CSF markers (leukocytes, glucose, protein and lactate) commonly used in routine care worldwide. The combination of LCN2, CSF leukocytes, glucose, protein and lactate resulted in the highest diagnostic performance for BM (area under the receiver operating characteristics curve, 0.96; 95% CI, 0.93-0.99). Data are available via ProteomeXchange with identifier PXD020510. CONCLUSIONS: LCN2 is a sensitive and specific biomarker for discriminating BM from a broad spectrum of other CNS infections. A prospective study is needed to assess the diagnostic utility of LCN2 in the diagnosis and management of CNS infections.

Three phylogenetic groups have driven the recent population expansion of Cryptococcus neoformans.

Cryptococcus neoformans (C. neoformans var. grubii) is an environmentally acquired pathogen causing 181,000 HIV-associated deaths each year. We sequenced 699 isolates, primarily C. neoformans from HIV-infected patients, from 5 countries in Asia and Africa. The phylogeny of C. neoformans reveals a recent exponential population expansion, consistent with the increase in the number of susceptible hosts. In our study population, this expansion has been driven by three sub-clades of the C. neoformans VNIa lineage; VNIa-4, VNIa-5 and VNIa-93. These three sub-clades account for 91% of clinical isolates sequenced in our study. Combining the genome data with clinical information, we find that the VNIa-93 sub-clade, the most common sub-clade in Uganda and Malawi, was associated with better outcomes than VNIa-4 and VNIa-5, which predominate in Southeast Asia. This study lays the foundation for further work investigating the dominance of VNIa-4, VNIa-5 and VNIa-93 and the association between lineage and clinical phenotype.