Combining supervised and unsupervised analyses to quantify behavioral phenotypes and validate therapeutic efficacy in a triple transgenic mouse model of Alzheimer's disease.

Behavioral testing is an essential tool for evaluating cognitive function and dysfunction in preclinical research models. This is of special importance in the study of neurological disorders such as Alzheimer's disease. However, the reproducibility of classic behavioral assays is frequently compromised by interstudy variation, leading to ambiguous conclusions about the behavioral markers characterizing the disease. Here, we identify age- and genotype-driven differences between 3xTg-AD and non-transgenic control mice using a low-cost, highly customizable behavioral assay that requires little human intervention. Through behavioral phenotyping combining both supervised and unsupervised behavioral classification methods, we are able to validate the preventative effects of the immunosuppressant cyclosporine A in a rodent model of Alzheimer's disease, as well as the partially ameliorating effects of candidate drugs nebivolol and cabozantinib.

A vagal reflex evoked by airway closure.

Airway integrity must be continuously maintained throughout life. Sensory neurons guard against airway obstruction and, on a moment-by-moment basis, enact vital reflexes to maintain respiratory function1,2. Decreased lung capacity is common and life-threatening across many respiratory diseases, and lung collapse can be acutely evoked by chest wall trauma, pneumothorax or airway compression. Here we characterize a neuronal reflex of the vagus nerve evoked by airway closure that leads to gasping. In vivo vagal ganglion imaging revealed dedicated sensory neurons that detect airway compression but not airway stretch. Vagal neurons expressing PVALB mediate airway closure responses and innervate clusters of lung epithelial cells called neuroepithelial bodies (NEBs). Stimulating NEBs or vagal PVALB neurons evoked gasping in the absence of airway threats, whereas ablating NEBs or vagal PVALB neurons eliminated gasping in response to airway closure. Single-cell RNA sequencing revealed that NEBs uniformly express the mechanoreceptor PIEZO2, and targeted knockout of Piezo2 in NEBs eliminated responses to airway closure. NEBs were dispensable for the Hering-Breuer inspiratory reflex, which indicated that discrete terminal structures detect airway closure and inflation. Similar to the involvement of Merkel cells in touch sensation3,4, NEBs are PIEZO2-expressing epithelial cells and, moreover, are crucial for an aspect of lung mechanosensation. These findings expand our understanding of neuronal diversity in the airways and reveal a dedicated vagal pathway that detects airway closure to help preserve respiratory function.

Personalized Tacrolimus Dosing After Liver Transplantation: A Randomized Clinical Trial.

Background: Inter- and intra-individual variability in tacrolimus dose requirements mandates empirical clinician-titrated dosing that frequently results in deviation from a narrow target range. Improved methods to individually dose tacrolimus are needed. Our objective was to determine whether a quantitative, dynamically-customized, phenotypic-outcome-guided dosing method termed Phenotypic Personalized Medicine (PPM) would improve target drug trough maintenance. Methods: In a single-center, randomized, pragmatic clinical trial ( NCT03527238 ), 62 adults were screened, enrolled, and randomized prior to liver transplantation 1:1 to standard-of-care (SOC) clinician-determined or PPM-guided dosing of tacrolimus. The primary outcome measure was percent days with large (>2 ng/mL) deviation from target range from transplant to discharge. Secondary outcomes included percent days outside-of-target-range and mean area-under-the-curve (AUC) outside-of-target-range per day. Safety measures included rejection, graft failure, death, infection, nephrotoxicity, or neurotoxicity. Results: 56 (29 SOC, 27 PPM) patients completed the study. The primary outcome measure was found to be significantly different between the two groups. Patients in the SOC group had a mean of 38.4% of post-transplant days with large deviations from target range; the PPM group had 24.3% of post-transplant days with large deviations; (difference -14.1%, 95% CI: -26.7 to -1.5 %, P=0.029). No significant differences were found in the secondary outcomes. In post-hoc analysis, the SOC group had a 50% longer median length-of-stay than the PPM group [15 days (Q1-Q3: 11-20) versus 10 days (Q1-Q3: 8.5-12); difference 5 days, 95% CI: 2-8 days, P=0.0026]. Conclusions: PPM guided tacrolimus dosing leads to better drug level maintenance than SOC. The PPM approach leads to actionable dosing recommendations on a day-to-day basis. Lay Summary: In a study on 62 adults who underwent liver transplantation, researchers investigated whether a new dosing method called Phenotypic Personalized Medicine (PPM) would improve daily dosing of the immunosuppression drug tacrolimus. They found that PPM guided tacrolimus dosing leads to better drug level maintenance than the standard-of-care clinician-determined dosing. This means that the PPM approach leads to actionable dosing recommendations on a day-to-day basis and can help improve patient outcomes.

An 8-cage imaging system for automated analyses of mouse behavior.

The analysis of mouse behavior is used in biomedical research to study brain function in health and disease. Well-established rapid assays allow for high-throughput analyses of behavior but have several drawbacks, including measurements of daytime behaviors in nocturnal animals, effects of animal handling, and the lack of an acclimation period in the testing apparatus. We developed a novel 8-cage imaging system, with animated visual stimuli, for automated analyses of mouse behavior in 22-h overnight recordings. Software for image analysis was developed in two open-source programs, ImageJ and DeepLabCut. The imaging system was tested using 4-5 month-old female wild-type mice and 3xTg-AD mice, a widely-used model to study Alzheimer's disease (AD). The overnight recordings provided measurements of multiple behaviors including acclimation to the novel cage environment, day and nighttime activity, stretch-attend postures, location in various cage areas, and habituation to animated visual stimuli. The behavioral profiles were different in wild-type and 3xTg-AD mice. AD-model mice displayed reduced acclimation to the novel cage environment, were hyperactive during the first hour of darkness, and spent less time at home in comparison to wild-type mice. We propose that the imaging system may be used to study various neurological and neurodegenerative disorders, including Alzheimer's disease.

Full-field strain mapping of healthy and pathological mouse aortas using stereo digital image correlation.

The murine aorta is a complex, heterogeneous structure that undergoes large and sometimes asymmetrical deformations under loading. For analytical convenience, mechanical behavior is predominantly described using global quantities that fail to capture critical local information essential to elucidating aortopathic processes. Here, in our methodological study, we used stereo digital image correlation (StereoDIC) to measure the strain profiles of speckle-patterned healthy and elastase-infused, pathological mouse aortas submerged in a temperature-controlled liquid medium. Our unique device rotates two 15-degree stereo-angle cameras that gather sequential digital images while simultaneously performing conventional biaxial pressure-diameter and force-length testing. A StereoDIC Variable Ray Origin (VRO) camera system model is employed to correct for high-magnification image refraction through hydrating physiological media. The resultant Green-Lagrange surface strain tensor was quantified at different blood vessel inflation pressures, axial extension ratios, and after aneurysm-initiating elastase exposure. Quantified results capture large, heterogeneous, inflation-related, circumferential strains that are drastically reduced in elastase-infused tissues. Shear strains, however, were very small on the tissue's surface. Spatially averaged StereoDIC-based strains were generally more detailed than those determined using conventional edge detection techniques.

Enteroendocrine cell lineages that differentially control feeding and gut motility.

Enteroendocrine cells are specialized sensory cells of the gut-brain axis that are sparsely distributed along the intestinal epithelium. The functions of enteroendocrine cells have classically been inferred by the gut hormones they release. However, individual enteroendocrine cells typically produce multiple, sometimes apparently opposing, gut hormones in combination, and some gut hormones are also produced elsewhere in the body. Here, we developed approaches involving intersectional genetics to enable selective access to enteroendocrine cells in vivo in mice. We targeted FlpO expression to the endogenous Villin1 locus (in Vil1-p2a-FlpO knock-in mice) to restrict reporter expression to intestinal epithelium. Combined use of Cre and Flp alleles effectively targeted major transcriptome-defined enteroendocrine cell lineages that produce serotonin, glucagon-like peptide 1, cholecystokinin, somatostatin, or glucose-dependent insulinotropic polypeptide. Chemogenetic activation of different enteroendocrine cell types variably impacted feeding behavior and gut motility. Defining the physiological roles of different enteroendocrine cell types provides an essential framework for understanding sensory biology of the intestine.

Increased TGFß1 and SMAD3 Contribute to Age-Related Aortic Valve Calcification.

Aims: Calcific aortic valve disease (CAVD) is a progressive heart disease that is particularly prevalent in elderly patients. The current treatment of CAVD is surgical valve replacement, but this is not a permanent solution, and it is very challenging for elderly patients. Thus, a pharmacological intervention for CAVD may be beneficial. In this study, we intended to rescue aortic valve (AV) calcification through inhibition of TGFß1 and SMAD3 signaling pathways. Methods and Results: The klotho gene, which was discovered as an aging-suppressor gene, has been observed to play a crucial role in AV calcification. The klotho knockout (Kl -/-) mice have shorter life span (8-12 weeks) and develop severe AV calcification. Here, we showed that increased TGFß1 and TGFß-dependent SMAD3 signaling were associated with AV calcification in Kl -/- mice. Next, we generated Tgfb1- and Smad3-haploinsufficient Kl -/- mice to determine the contribution of TGFß1 and SMAD3 to the AV calcification in Kl -/- mice. The histological and morphometric evaluation suggested a significant reduction of AV calcification in Kl -/-; Tgfb1 ± mice compared to Kl -/- mice. Smad3 heterozygous deletion was observed to be more potent in reducing AV calcification in Kl -/- mice compared to the Kl -/-; Tgfb1 ± mice. We observed significant inhibition of Tgfb1, Pai1, Bmp2, Alk2, Spp1, and Runx2 mRNA expression in Kl -/-; Tgfb1 ± and Kl -/-; Smad3 ± mice compared to Kl -/- mice. Western blot analysis confirmed that the inhibition of TGFß canonical and non-canonical signaling pathways were associated with the rescue of AV calcification of both Kl -/-; Tgfb1 ± and Kl -/-; Smad3 ± mice. Conclusion: Overall, inhibition of the TGFß1-dependent SMAD3 signaling pathway significantly blocks the development of AV calcification in Kl -/- mice. This information is useful in understanding the signaling mechanisms involved in CAVD.

Integrating central nervous system metagenomics and host response for diagnosis of tuberculosis meningitis and its mimics.

The epidemiology of infectious causes of meningitis in sub-Saharan Africa is not well understood, and a common cause of meningitis in this region, Mycobacterium tuberculosis (TB), is notoriously hard to diagnose. Here we show that integrating cerebrospinal fluid (CSF) metagenomic next-generation sequencing (mNGS) with a host gene expression-based machine learning classifier (MLC) enhances diagnostic accuracy for TB meningitis (TBM) and its mimics. 368 HIV-infected Ugandan adults with subacute meningitis were prospectively enrolled. Total RNA and DNA CSF mNGS libraries were sequenced to identify meningitis pathogens. In parallel, a CSF host transcriptomic MLC to distinguish between TBM and other infections was trained and then evaluated in a blinded fashion on an independent dataset. mNGS identifies an array of infectious TBM mimics (and co-infections), including emerging, treatable, and vaccine-preventable pathogens including Wesselsbron virus, Toxoplasma gondii, Streptococcus pneumoniae, Nocardia brasiliensis, measles virus and cytomegalovirus. By leveraging the specificity of mNGS and the sensitivity of an MLC created from CSF host transcriptomes, the combined assay has high sensitivity (88.9%) and specificity (86.7%) for the detection of TBM and its many mimics. Furthermore, we achieve comparable combined assay performance at sequencing depths more amenable to performing diagnostic mNGS in low resource settings.

Olfactory landmarks and path integration converge to form a cognitive spatial map.

The convergence of internal path integration and external sensory landmarks generates a cognitive spatial map in the hippocampus. We studied how localized odor cues are recognized as landmarks by recording the activity of neurons in CA1 during a virtual navigation task. We found that odor cues enriched place cell representations, dramatically improving navigation. Presentation of the same odor at different locations generated distinct place cell representations. An odor cue at a proximal location enhanced the local place cell density and also led to the formation of place cells beyond the cue. This resulted in the recognition of a second, more distal odor cue as a distinct landmark, suggesting an iterative mechanism for extending spatial representations into unknown territory. Our results establish that odors can serve as landmarks, motivating a model in which path integration and odor landmarks interact sequentially and iteratively to generate cognitive spatial maps over long distances.

Osteoclast-Mediated Cell Therapy as an Attempt to Treat Elastin Specific Vascular Calcification.

Inflammation and stiffness in the arteries is referred to as vascular calcification. This process is a prevalent yet poorly understood consequence of cardiovascular disease and diabetes mellitus, comorbidities with few treatments clinically available. Because this is an active process similar to bone formation, it is hypothesized that osteoclasts (OCs), bone-resorbing cells in the body, could potentially work to reverse existing calcification by resorbing bone material. The receptor activator of nuclear kappa B-ligand (RANKL) is a molecule responsible for triggering a response in monocytes and macrophages that allows them to differentiate into functional OCs. In this study, OC and RANKL delivery were employed to determine whether calcification could be attenuated. OCs were either delivered via direct injection, collagen/alginate microbeads, or collagen gel application, while RANKL was delivered via injection, through either a porcine subdermal model or aortic injury model. While in vitro results yielded a decrease in calcification using OC therapy, in vivo delivery mechanisms did not provide control or regulation to keep cells localized long enough to induce calcification reduction. However, these results do provide context and direction for the future of OC therapy, revealing necessary steps for this treatment to effectively reduce calcification in vivo. The discrepancy between in vivo and in vitro success for OC therapy points to the need for a more stable and time-controlled delivery mechanism that will allow OCs not only to remain at the site of calcification, but also to be regulated so that they are healthy and functioning normally when introduced to diseased tissue.

HFIP promoted thio(hetero)arylation of imidazoheterocycles under metal- and base-free conditions.

A new reaction methodology has been developed for HFIP promoted Thio(hetero)arylation of imidazoheterocycles under metal and base-free conditions. To the best of our knowledge, this is the first report that describes linking of imidazopyridines with electron deficient heteroarenes through a sulphur atom and also for the synthesis of most of these compounds. The reaction conditions are well tolerated by almost all kinds of 2-chloroheteroarenes and a wide range of imidazoheterocycles. The synthesized compounds can show significant biological properties.

Genome-Wide Analysis of Heat Shock Transcription Factors in Ziziphus jujuba Identifies Potential Candidates for Crop Improvement Under Abiotic Stress.

Plant heat shock transcription factors (Hsfs) play a significant role in adoption under abiotic stress conditions by modulating the expression of several stress-responsive genes. Analysis of the Hsf gene family will serve to understand the molecular mechanism which is involved in response to abiotic stress. The Ziziphus species grows in warm and dry regions and is inherently tolerant to abiotic stress conditions; thus, Ziziphus is a highly enriched source of genes conferring abiotic stress tolerance. Therefore, the present study provides a comprehensive genome-wide analysis of the Hsf gene family in Z. jujuba. Identified 21 non-redundant Hsf genes were grouped into three major classes (classes A, B, and C) based on the phylogenetic analysis. Promoter and gene ontology analysis suggested that ZjHsfs perform diverse functions in response to abiotic stress conditions. Two paralogous pairs resulting from tandem gene duplication events were identified. Also, physio-chemical properties of chromosomal locations, gene structure, motifs, and protein domain organization of Hsfs were analyzed. Real-time PCR expression analyses revealed that most of the Z. jujuba Hsf genes are differentially expressed in response to heat stress. The analysis suggested ZjHsf-2, ZjHsf-3, ZjHsf-5, ZjHsf-7, ZjHsf-8, ZjHsf-10, ZjHsf-12, ZjHsf-17, and ZjHsf-18 were the outstanding candidate genes for imparting heat stress tolerance and for future functional analysis. The present analysis laid the foundation for understanding the molecular mechanism of the Hsf gene family regulating Z. jujuba development and tolerance to abiotic stress conditions.

Genome-wide analysis of Hsp70 and Hsp100 gene families in Ziziphus jujuba.

The Ziziphus species are naturally tolerant to a range of abiotic stresses. Therefore, it is expected that they are an enriched source of genes conferring stress tolerance. Heat shock proteins (Hsps) play a significant role in plants in imparting tolerance against abiotic stress conditions. To get an insight into potential Hsp function in Ziziphus, we performed a genome-wide analysis and expression study of Hsp70 and Hsp100 gene families in Ziziphus jujuba. We identified 21 and 6 genes of the ZjHsp70 and ZjHsp100 families, respectively. Physiochemical properties, chromosomal location, gene structure, motifs, and protein domain organization were analysed for structural and functional characterization. We identified the contribution of tandem and segmental gene duplications in expansions of ZjHsp70s and ZjHsp100s in Z. jujuba. Promoter analysis suggested that ZjHsp70s and ZjHsp100s perform diverse functions related to abiotic stress. Furthermore, expression analyses revealed that most of the Z. jujuba Hsp genes are differentially expressed in response to heat, drought, and salinity stress. Our analyses suggested ZjHsp70-3, ZjHsp70-5, ZjHsp70-6, ZjHsp70-16, ZjHsp70-17, ZjHsp70-20, ZjHsp100-1, ZjHsp100-2, and ZjHsp100-3 are potential candidates for further functional analysis and with regard to breeding new more resilient strains. The present analysis laid the foundation for understanding the molecular mechanism of Hsps70 and Hsp100 gene families regulating abiotic stress tolerance in Z. jujuba.

Portosystemic shunts versus endoscopic intervention with or without medical treatment for prevention of rebleeding in people with cirrhosis.

BACKGROUND: People with liver cirrhosis who have had one episode of variceal bleeding are at risk for repeated episodes of bleeding. Endoscopic intervention and portosystemic shunts are used to prevent further bleeding, but there is no consensus as to which approach is preferable. OBJECTIVES: To compare the benefits and harms of shunts (surgical shunts (total shunt (TS), distal splenorenal shunt (DSRS), or transjugular intrahepatic portosystemic shunt (TIPS)) versus endoscopic intervention (endoscopic sclerotherapy or banding, or both) with or without medical treatment (non-selective beta blockers or nitrates, or both) for prevention of variceal rebleeding in people with liver cirrhosis. SEARCH METHODS: We searched the CHBG Controlled Trials Register; CENTRAL, in the Cochrane Library; MEDLINE Ovid; Embase Ovid; LILACS (Bireme); Science Citation Index - Expanded (Web of Science); and Conference Proceedings Citation Index - Science (Web of Science); as well as conference proceedings and the references of trials identified until 22 June 2020. We contacted study investigators and industry researchers. SELECTION CRITERIA: Randomised clinical trials comparing shunts versus endoscopic interventions with or without medical treatment in people with cirrhosis who had recovered from a variceal haemorrhage. DATA COLLECTION AND ANALYSIS: We used standard methodological procedures expected by Cochrane. When possible, we collected data to allow intention-to-treat analysis. For each outcome, we estimated a meta-analysed estimate of treatment effect across trials (risk ratio for binary outcomes). We used random-effects model meta-analysis as our main analysis and as a means of presenting results. We reported differences in means for continuous outcomes without a meta-analytic estimate due to high variability in their assessment among all trials. We assessed the certainty of evidence using GRADE. MAIN RESULTS: We identified 27 randomised trials with 1828 participants. Three trials assessed TSs, five assessed DSRSs, and 19 trials assessed TIPSs. The endoscopic intervention was sclerotherapy in 16 trials, band ligation in eight trials, and a combination of band ligation and either sclerotherapy or glue injection in three trials. In eight trials, endoscopy was combined with beta blockers (in one trial plus isosorbide mononitrate). We judged all trials to be at high risk of bias. We assessed the certainty of evidence for all the outcome review results as very low (i.e. the true effects of the results are likely to be substantially different from the results of estimated effects). The very low evidence grading is due to the overall high risk of bias for all trials, and to imprecision and publication bias for some outcomes. Therefore, we are very uncertain whether portosystemic shunts versus endoscopy interventions with or without medical treatment have effects on all-cause mortality (RR 0.99, 95% CI 0.86 to 1.13; 1828 participants; 27 trials), on rebleeding (RR 0.40, 95% CI 0.33 to 0.50; 1769 participants; 26 trials), on mortality due to rebleeding (RR 0.51, 95% CI 0.34 to 0.76; 1779 participants; 26 trials), and on occurrence of hepatic encephalopathy, both acute (RR 1.60, 95% CI 1.33 to 1.92; 1649 participants; 24 trials) and chronic (RR 2.51, 95% CI 1.38 to 4.55; 956 participants; 13 trials). No data were available regarding health-related quality of life. Analysing each modality of portosystemic shunts individually (i.e. TS, DSRS, and TIPS) versus endoscopic interventions with or without medical treatment, we are very uncertain if each type of shunt has effect on all-cause mortality: TS, RR 0.46, 95% CI 0.19 to 1.13; 164 participants; 3 trials; DSRS, RR 0.93, 95% CI 0.65 to 1.33; 352 participants; 4 trials; and TIPS, RR 1.10, 95% CI 0.92 to 1.31; 1312 participants; 19 trial; on rebleeding: TS, RR 0.28, 95% CI 0.14 to 0.56; 127 participants; 2 trials; DSRS, RR 0.26, 95% CI 0.11 to 0.65; 330 participants; 5 trials; and TIPS, RR 0.44, 95% CI 0.36 to 0.55; 1312 participants; 19 trials; on mortality due to rebleeding: TS, RR 0.25, 95% CI 0.06 to 0.96; 164 participants; 3 trials; DSRS, RR 0.31, 95% CI 0.13 to 0.74; 352 participants; 5 trials; and TIPS, RR 0.65, 95% CI 0.40 to 1.04; 1263 participants; 18 trials; on acute hepatic encephalopathy: TS, RR 1.66, 95% CI 0.70 to 3.92; 115 participants; 2 trials; DSRS, RR 1.70, 95% CI 0.94 to 3.08; 287 participants; 4 trials, TIPS, RR 1.61, 95% CI 1.29 to 1.99; 1247 participants; 18 trials; and chronic hepatic encephalopathy: TS, Fisher's exact test P = 0.11; 69 participants; 1 trial; DSRS, RR 4.87, 95% CI 1.46 to 16.23; 170 participants; 2 trials; and TIPS, RR 1.88, 95% CI 0.93 to 3.80; 717 participants; 10 trials. The proportion of participants with shunt occlusion or dysfunction was overall 37% (95% CI 33% to 40%). It was 3% (95% CI 0.8% to 10%) following TS, 7% (95% CI 3% to 13%) following DSRS, and 47.1% (95% CI 43% to 51%) following TIPS. Shunt dysfunction in trials utilising polytetrafluoroethylene-covered stents was 17% (95% CI 11% to 24%). Length of inpatient hospital stay and cost were not comparable across trials. Funding was unclear in 16 trials; 11 trials were funded by government, local hospitals, or universities. AUTHORS' CONCLUSIONS: Evidence on whether portosystemic shunts versus endoscopy interventions with or without medical treatment in people with cirrhosis and previous hypertensive portal bleeding have little or no effect on all-cause mortality is very uncertain. Evidence on whether portosystemic shunts may reduce bleeding and mortality due to bleeding while increasing hepatic encephalopathy is also very uncertain. We need properly conducted trials to assess effects of these interventions not only on assessed outcomes, but also on quality of life, costs, and length of hospital stay.

Functional Applications of Polyarginine-Hyaluronic Acid-Based Electrostatic Complexes.

Background: Electrostatic complexes of poly (l-Arginine) (pArg) and hyaluronic acid (HA) have been investigated for their functional applications to supply free or polymeric form of l-Arginine (Arg) to target cells. As a vital amino acid, Arg plays significant role in multitude of pathophysiological processes ranging from wound healing to cancer. However, serum arginase expression and toxicity of Arg at cellular level renders exogenous delivery of this amino acid a challenging task. We showed that polyarginine-hyaluronic acid ionic nanocomplexes (pArg-HA iNCs) could be an effective way to deliver Arg to target cell populations. Materials and Methods: These electrostatic complexes were prepared by mixing HA (average m.w. of 200 kDa) with pArg (m.w. 5-15 kDa; Sigma) in aqueous solutions and purifying over glycerol. Nanocomplexes were characterized for their particle size, surface charge, capacity to release l-Arg, and intracellular uptake of complexes. Results: Synthesized nanocomplexes showed hydrodynamic diameter ranging from 140-306 nm depending on the content of pArg or HA within the formulation. With surface charge (ζ-potential) of -29 mV, the nanocomplexes showed pH-dependent release of Arg. At pH 7.4, pArg-HA iNCs released 30% of the total Arg-content, while at pH 5.0, 60% of Arg was released after 24 h. These electrostatically stabilized complexes were found to promote growth of human dermal fibroblasts (HDF) in wound-healing assay and increased nitric oxide (NO) activity in these cells in a time-dependent manner. Nanocomplexes also showed cellular uptake and enhanced dose-dependent toxicity against two pancreatic cancer cell lines, i.e. MIA PaCa-2 and Panc-1. Interestingly, the cytotoxic effect was synergized upon pre-treatment of the cells with a frontline chemotherapeutic agent, gemcitabine (GEM), and was not observed when the cells were treated with Arg alone. Conclusion: As such, this communication shows the prospect of pArg-HA iNC electrostatic nanocomplexes to interact and interfere with intracellular Arg metabolic machinery conducive to rescuing different pathological conditions.

Electrospun nanofibers in cancer research: from engineering of in vitro 3D cancer models to therapy.

Electrospinning is historically related to tissue engineering due to its ability to produce nano-/microscale fibrous materials with mechanical and functional properties that are extremely similar to those of the extracellular matrix of living tissues. The general interest in electrospun fibrous matrices has recently expanded to cancer research both as scaffolds for in vitro cancer modelling and as patches for in vivo therapeutic delivery. In this review, we examine electrospinning by providing a brief description of the process and overview of most materials used in this process, discussing the effect of changing the process parameters on fiber conformations and assemblies. Then, we describe two different applications of electrospinning in service of cancer research: firstly, as three-dimensional (3D) fibrous materials for generating in vitro pre-clinical cancer models; and secondly, as patches encapsulating anticancer agents for in vivo delivery.

Null strain analysis of submerged aneurysm analogues using a novel 3D stereomicroscopy device.

To measure the inhomogeneous 3D-strain fields present during inflation-extension testing of physiologically submerged micro-aneurysms, a Stereo Digital Image Correlation (StereoDIC) microscopy system is developed that revolves 15° stereo-angle cameras around a centrally-mounted target. Calibration is performed using submerged dot patterns and system accuracy verified using strain and deformation analyses for rigid body motions of speckle-patterned, micro-aneurysmal surrogates. In terms of the Green-Lagrange strain tensor and the 3D displacement fields, the results are stable even after 120 minutes, with maxima in both strain bias and strain standard deviation less than 2E-03 for all components, and micron-level displacement standard deviation.

Cell deformation and acquired drug resistance: elucidating the major influence of drug-nanocarrier delivery systems.

Cancer diagnosis and its stage-wise assessment are determined through invasive solid tissue biopsies. Conversely, cancer imaging is enriched through emission tomography and longitudinal high-resolution analysis for the early detection of cancer through altered cell morphology and cell-deformation. Similarly, in post multiple chemo-cycle exposures, the tumor regression and progression thereafter are not well understood. Here, we report chemo-cycles of doxorubicin (Dox) carrying nanoparticles (NPs) to be highly indicative of cell deformation and a progressive indicator of phenotypic expressions of acquired drug resistance (ADR). We designed graphene (G) based nanocarriers by chemically conjugating multiple components: (i) G; (ii) iron oxide (Fe3O4) NPs; and (iii) Dox through a cysteine (Cys) linker (G-Dox and G-Cys-Fe3O4-Dox). Although Dox underwent cell diffusion, the G-based nanocarriers followed a receptor-mediated endocytosis which created a profound impact on the cell membrane integrity. ADR owing to Dox and G-based nanocarriers was analyzed through a cytotoxicity assay, cell morphology deformation parameters and cellular uptake kinetic patterns. Interestingly, after the third chemo-cycle, G-Dox incubated cells showed the greatest decrease in the alteration of the nuclear surface area (NSA) of ∼28%, a ∼40% reduction of the cell surface area (CSA) and a ∼32% increase in the cell roundness (CRd). Our results suggested that the G-based nanocarriers induced the cell deformation process, subsequently resulting in ADR. Although the G-based nanocarriers initiated ADR, G-Dox was most cytotoxic to cancer cells and induced the maximum cell morphology deformation within our scope of study. This outcome implies caution is needed when using G-based nanocarriers and other multi-component nanosystems for Dox delivery as they lead to possible phenotypic expressions of drug resistance in cancer cells.

Cellular regeneration and proliferation on polymeric 3D inverse-space substrates and the effect of doxorubicin.

Spatial arrangement for cells and the opportunity thereof have implications in cell regeneration and cell proliferation. 3D inverse space (3DIS) substrates with micron-sized pores are fabricated under controlled environmental conditions from polymers such as poly(lactic-co-glycolic) acid (PLGA), poly(lactic acid) (PLA) and poly(styrene) (PS). The characterization of 3DIS substrates by optical microscopy, scanning probe microscopy (SPM), etc. shows pores within 1-18 µm diameter and prominent surface roughness extending up to 3.9 nm in height over its base. Conversely, to compare two-dimensional (2D) versus 3DIS substrates, the crucial variables of cell height, cell spreading area and cell volume are compared using lung adenocarcinoma (A549) cells. The results indicate an average cell thickness of ∼6 µm on a glass substrate whereas cells on PLGA 3DIS were ∼12 µm in height, occasionally reaching 20 µm, with a 40% decreased cell spreading area. A549 cells cultured on polymer 3DIS substrates show a cell regeneration growth pattern, dependent on the available spatial volume. Furthermore, PLGA 3DIS cell culture systems with and without graded doxorubicin (DOX) pre-treatment result in potent cell inhibition and cell proliferation, respectively. Additionally, standard DOX administration to A549 cells in the PLGA 3DIS system revealed altered drug sensitivity. 3DIS demonstrates utility in facilitating cellular regeneration and mimicking cell proliferation in defined spatial arrangements.

Arterial Baroreceptors Sense Blood Pressure through Decorated Aortic Claws.

Mechanosensory neurons across physiological systems sense force using diverse terminal morphologies. Arterial baroreceptors are sensory neurons that monitor blood pressure for real-time stabilization of cardiovascular output. Various aortic sensory terminals have been described, but those that sense blood pressure are unclear because of a lack of selective genetic tools. Here, we find that all baroreceptor neurons are marked in Piezo2-ires-Cre mice and then use genetic approaches to visualize the architecture of mechanosensory endings. Cre-guided ablation of vagal and glossopharyngeal PIEZO2 neurons eliminates the baroreceptor reflex and aortic depressor nerve effects on blood pressure and heart rate. Genetic mapping reveals that PIEZO2 neurons form a distinctive mechanosensory structure: macroscopic claws that surround the aortic arch and exude fine end-net endings. Other arterial sensory neurons that form flower-spray terminals are dispensable for baroreception. Together, these findings provide structural insights into how blood pressure is sensed in the aortic vessel wall.