Racial and ethnic disparities in gallbladder cancer: A two-decade analysis of incidence and mortality rates in the US.

BACKGROUND: Gallbladder cancer (GBC) is an aggressive malignancy that is usually diagnosed at a late stage. Prior data showed increasing incidence of GBC in the US. However, little is known about race/ethnic-specific incidence and mortality trends of GBC per stage at diagnosis. Therefore, we aimed to conduct a time-trend analysis of GBC incidence and mortality rates categorized by race/ethnicity and stage-at-diagnosis. METHODS: Age-adjusted GBC incidence and mortality rates were calculated using SEER*Stat software from the United States Cancer Statistics database (covers ~98% of US population between 2001 and 2020) and NCHS (covers ~100% of the US population between 2000 and 2020) databases, respectively. Race/Ethnic groups were Non-Hispanic-White (NHW), Non-Hispanic-Black (NHB), Hispanic, Non-Hispanic-Asian/Pacific-Islander (NHAPI), and Non-Hispanic-American-Indian/Alaska-Native (NHAIAN). Stage-at-diagnoses were all stages, early, regional, and distant stages. Joinpoint regression was used to generate time-trends [annual percentage change (APC) and average APC (AAPC)] with parametric estimations and a two-sided t-test (p-value cut-off 0.05). RESULTS: 76,873 patients were diagnosed with GBC with decreasing incidence rates in all races/ethnicities except NHB who experienced an increasing trend between 2001 and 2014 (APC = 2.08, p < 0.01) and plateauing afterward (APC = -1.21, p = 0.31); (AAPC = 1.03, p = 0.03). Among early-stage tumors (9927 patients), incidence rates were decreasing only in Hispanic (AAPC = -4.24, p = 0.006) while stable in other races/ethnicities (NHW: AAPC = -2.61, p = 0.39; NHB: AAPC = -1.73, p = 0.36). For regional-stage tumors (29,690 patients), GBC incidence rates were decreasing only in NHW (AAPC = -1.61, p < 0.001) while stable in other races/ethnicities (NHB: AAPC = 0.73, p = 0.34; Hispanic: AAPC = -1.58, p = 0.24; NHAPI: AAPC = -1.22, p = 0.07). For distant-stage tumors (31,735 patients), incidence rates were increasing in NHB (AAPC = 2.72, p < 0.001), decreasing in Hispanic (AAPC = -0.64, p = 0.04), and stable in NHW (AAPC = 0.07, p = 0.84) and NHAPI (AAPC = 0.79, p = 0.13). There were 43,411 deaths attributed to GBC with decreasing mortality rates in all races/ethnicities except NHB who experienced a stable trend (AAPC = 0.25, p = 0.25). CONCLUSION: Nationwide data over the last two decades show that NHB patients experienced increasing GBC incidence between 2001 and 2014 followed by stabilization of the rates. This increase was driven by late-stage tumors and occurred in the first decade. NHB also experienced non-improving GBC mortality, compared to other race and ethnic groups who had decreasing mortality. This can be due to lack of timely-access to healthcare leading to delayed diagnosis and worse outcomes. Future studies are warranted to investigate contributions to the revealed racial and ethnic disparities, especially in NHB, to improve early detection.

Treating Helicobacter pylori and Recurrent Clostridioides difficile Coinfection: A Delicate Balance in Management and a Need for Guidelines.

Treating Helicobacter pylori and Clostridioides difficile coinfection presents a challenging clinical dilemma. Treating H. pylori may increase the risk of C. difficile, and antibiotics generally have been shown to increase the risk of C. difficile infection/recurrence. While it may be reasonable to delay H. pylori treatment, this is especially challenging when there is an acute indication to treat H. pylori such as peptic ulceration or bleeding. There are no guidelines on the management of H. pylori and C. difficile coinfection. We report a patient who had H. pylori and recurrent C. difficile coinfection and suggest a management algorithm based on literature review and our institutional experience. Our patient received quadruple therapy for H. pylori along with vancomycin prophylaxis, taper, and a dose of bezlotoxumab and experienced good outcomes with resolution of his gastrointestinal bleeding and diarrhea.

Geographical Variations in Early Onset Colorectal Cancer in the United States between 2001 and 2020.

BACKGROUND: Colorectal cancer remains the second leading cause of cancer-related death in the US. As early-onset colorectal cancer (EO-CRC) becomes more prevalent in the US, research attention has shifted towards identifying at-risk populations. Previous studies have highlighted the rising rate of early-onset adenocarcinoma (ADC) and neuroendocrine tumors (NET) in the US. However, data on geographical variations of EO-CRC are scarce. Hence, our study aims to analyze time trends in EO-CRC incidence rates across various US regions and to assess these trends by sex and histopathological subtypes (ADC and NET). METHODS: We analyze data spanning from 2001 to 2020 from the United States Cancer Statistics (USCS) database, covering nearly 98% of the US population. Using SEER*Stat software version (8.4.2, NCI), we calculated EO-CRC incidence rates among adults aged 20-54 years, adjusting for the age standard 2000 US population. The rates were categorized by sex and US geographical regions into west, midwest, northeast, and south. Time trends, reported as annual percentage change (APC) and average APC (AAPC), were generated via Joinpoint Regression software (v.5.0.2, NCI) utilizing the weighted Bayesian Information Criteria "BIC" method to generate the best-fit trends with a two-sided p-value cutoff at 0.05. The rates were also stratified by histopathology into ADC and NET. RESULTS: Between 2001 and 2020, a total of 514,875 individuals were diagnosed with early-onset CRC in the US, with 54.78% being men. Incidence rates and trends varied across geographical regions. In the western region (comprising 106,685 patients, 54.85% men), incidence rates significantly increased in both women (AAPC = 1.37, p < 0.001) and men (AAPC = 1.34, p < 0.001). Similarly, in the midwestern region (with 110,380 patients, 55.46% men), there were significant increases in incidence rates among women (AAPC = 1.06, p < 0.001) and men (AAPC = 1.35, p < 0.001). The northeastern region (with 94,758 patients, 54.53% men) also witnessed significant increases in incidence rates for both women (AAPC = 0.71, p < 0.001) and men (AAPC = 0.84, p < 0.001). In contrast, the southern region (with 203,052 patients, 54.48% men) experienced slower increases in incidence rates among both women and men (AAPC = 0.25, p < 0.05 in women; AAPC = 0.66, p < 0.05 in men). When stratified by histopathology, incidence rates for adenocarcinomas (ADC) increased in all regions, most notably in the west (AAPC = 1.45, p < 0.05), and least in the south (AAPC = 0.46, p < 0.05). Conversely, for neuroendocrine tumors (NET), while incidence rates increased similarly across all regions, the pace was notably faster compared to ADC, particularly in the west (AAPC = 3.26, p < 0.05) and slower in the south (AAPC = 2.24, p < 0.05) Discussion: Our analysis of nationwide US data spanning two decades and encompassing over half a million early-onset CRC patients, representing nearly 98% of the US population, highlights significant temporal variation in incidence rates across various geographical regions. The most substantial increases in incidence rates were observed in the west, while the least pronounced changes were noted in the south, affecting both men and women. These trends persisted across the main CRC histopathological subtypes, with NET exhibiting a notably swifter pace of increase compared with ADC. These findings hold important implications for public health strategies and underscore the need for targeted interventions to address the rising burden of early-onset CRC across different regions in the US.

Industrial perspectives for personalized microneedles.

Microneedles and, subsequently, microneedle arrays are emerging miniaturized medical devices for painless transdermal drug delivery. New and improved additive manufacturing methods enable novel microneedle designs to be realized for preclinical and clinical trial assessments. However, current literature reviews suggest that industrial manufacturers and researchers have focused their efforts on one-size-fits-all designs for transdermal drug delivery, regardless of patient demographic and injection site. In this perspective article, we briefly review current microneedle designs, microfabrication methods, and industrialization strategies. We also provide an outlook where microneedles may become personalized according to a patient's demographic in order to increase drug delivery efficiency and reduce healing times for patient-centric care.

Two-Photon Polymerized Shape Memory Microfibers: A New Mechanical Characterization Method in Liquid.

Two-photon polymerization (TPP) has been widely used to create 3D micro- and nanoscale scaffolds for biological and mechanobiological studies, which often require the mechanical characterization of the TPP fabricated structures. To satisfy physiological requirements, most of the mechanical characterizations need to be conducted in liquid. However, previous characterizations of TPP fabricated structures were all conducted in air due to the limitation of conventional micro- and nanoscale mechanical testing methods. In this study, we report a new experimental method for testing the mechanical properties of TPP-printed microfibers in liquid. The experiments show that the mechanical behaviors of the microfibers tested in liquid are significantly different from those tested in air. By controlling the TPP writing parameters, the mechanical properties of the microfibers can be tailored over a wide range to meet a variety of mechanobiology applications. In addition, it is found that, in water, the plasticly deformed microfibers can return to their pre-deformed shape after tensile strain is released. The shape recovery time is dependent on the size of microfibers. The experimental method represents a significant advancement in mechanical testing of TPP fabricated structures and may help release the full potential of TPP fabricated 3D tissue scaffold for mechanobiological studies.

MHC class II-restricted antigen presentation is required to prevent dysfunction of cytotoxic T cells by blood-borne myeloids in brain tumors.

Cancer immunotherapy critically depends on fitness of cytotoxic and helper T cell responses. Dysfunctional cytotoxic T cell states in the tumor microenvironment (TME) are a major cause of resistance to immunotherapy. Intratumoral myeloid cells, particularly blood-borne myeloids (bbm), are key drivers of T cell dysfunction in the TME. We show here that major histocompatibility complex class II (MHCII)-restricted antigen presentation on bbm is essential to control the growth of brain tumors. Loss of MHCII on bbm drives dysfunctional intratumoral tumor-reactive CD8+ T cell states through increased chromatin accessibility and expression of Tox, a critical regulator of T cell exhaustion. Mechanistically, MHCII-dependent activation of CD4+ T cells restricts myeloid-derived osteopontin that triggers a chronic activation of NFAT2 in tumor-reactive CD8+ T cells. In summary, we provide evidence that MHCII-restricted antigen presentation on bbm is a key mechanism to directly maintain functional cytotoxic T cell states in brain tumors.

Oral Rabies Vaccine Strain SPBN GASGAS: Genetic Stability after Serial In Vitro and In Vivo Passaging.

Oral vaccination of wildlife has shown to be a very effective management tool in rabies control. Evaluation of the genetic stability of vaccine viruses before distributing vaccine baits in the environment is essential because all available oral rabies vaccines, including the genetically engineered rabies virus vaccine strain SPBN GASGAS (Rabitec), are based on replication-competent viruses. To evaluate the genetic stability of this vaccine strain, five serial passages of the Master Seed Virus (MSV) in the production cell line BHK21 Cl13 were performed. Furthermore, to test possible reversion to virulence, a back-passage study in suckling mouse brain (SMB) was performed. Subsequently, the pooled 5th SMB passage was inoculated intracerebrally (i.c.) in adult and suckling mice. The full genome sequences of the isolated 5th passage, in vivo and in vitro, were compared at both the consensus and the quasispecies level with the MSV. Additionally, the full genome sequence of the 6th SMB passage from the individual animals was determined and compared. Full-length integration of the double glycoprotein and modified base substitutions at amino acid position 194 and 333 of the glycoprotein could be verified in all 5th and 6th passage samples. Overall, 11 single nucleotide polymorphisms (SNPs) were detected in the 5th pooled SMB passage, 4 with frequency between 10 and 20%, and 7 with between 2.5 and 10%. SNPs that resulted in amino acid exchange were found in genes: N (one SNP), G (four SNPs), and L (three SNPs). However, none of these SNPs were associated with reversion to virulence since all adult mice inoculated i.c. with this material survived. In the individual samples of the 6th SMB passage 24 additional SNPs (>2.5%) were found, of which only 1 SNP (L-gene, position 6969) had a prevalence of >50% in 3 of 17 samples. The obtained results confirmed the stable expression of genetic modifications and the genetic stability of the consensus strain after serial in vivo and in vitro passaging.

LABRADOR-A Computational Workflow for Virus Detection in High-Throughput Sequencing Data.

High-throughput sequencing (HTS) allows detection of known and unknown viruses in samples of broad origin. This makes HTS a perfect technology to determine whether or not the biological products, such as vaccines are free from the adventitious agents, which could support or replace extensive testing using various in vitro and in vivo assays. Due to bioinformatics complexities, there is a need for standardized and reliable methods to manage HTS generated data in this field. Thus, we developed LABRADOR-an analysis pipeline for adventitious virus detection. The pipeline consists of several third-party programs and is divided into two major parts: (i) direct reads classification based on the comparison of characteristic profiles between reads and sequences deposited in the database supported with alignment of to the best matching reference sequence and (ii) de novo assembly of contigs and their classification on nucleotide and amino acid levels. To meet the requirements published in guidelines for biologicals' safety we generated a custom nucleotide database with viral sequences. We tested our pipeline on publicly available HTS datasets and showed that LABRADOR can reliably detect viruses in mixtures of model viruses, vaccines and clinical samples.

Geometrically defined environments direct cell division rate and subcellular YAP localization in single mouse embryonic stem cells.

Mechanotransduction via yes-associated protein (YAP) is a central mechanism for decision-making in mouse embryonic stem cells (mESCs). Nuclear localization of YAP is tightly connected to pluripotency and increases the cell division rate (CDR). How the geometry of the extracellular environment influences mechanotransduction, thereby YAP localization, and decision-making of single isolated mESCs is largely unknown. To investigate this relation, we produced well-defined 2D and 2.5D microenvironments and monitored CDR and subcellular YAP localization in single mESCs hence excluding cell-cell interactions. By systematically varying size and shape of the 2D and 2.5D substrates we observed that the geometry of the growth environment affects the CDR. Whereas CDR increases with increasing adhesive area in 2D, CDR is highest in small 2.5D micro-wells. Here, mESCs attach to all four walls and exhibit a cross-shaped cell and nuclear morphology. This observation indicates that changes in cell shape are linked to a high CDR. Inhibition of actomyosin activity abrogate these effects. Correspondingly, nuclear YAP localization decreases in inhibitor treated cells, suggesting a relation between cell shape, intracellular forces, and cell division rate. The simplicity of our system guarantees high standardization and reproducibility for monitoring stem cell reactions and allows addressing a variety of fundamental biological questions on a single cell level.

Mechanical stimulation of single cells by reversible host-guest interactions in 3D microscaffolds.

Many essential cellular processes are regulated by mechanical properties of their microenvironment. Here, we introduce stimuli-responsive composite scaffolds fabricated by three-dimensional (3D) laser lithography to simultaneously stretch large numbers of single cells in tailored 3D microenvironments. The key material is a stimuli-responsive photoresist containing cross-links formed by noncovalent, directional interactions between ß-cyclodextrin (host) and adamantane (guest). This allows reversible actuation under physiological conditions by application of soluble competitive guests. Cells adhering in these scaffolds build up initial traction forces of ~80 nN. After application of an equibiaxial stretch of up to 25%, cells remodel their actin cytoskeleton, double their traction forces, and equilibrate at a new dynamic set point within 30 min. When the stretch is released, traction forces gradually decrease until the initial set point is retrieved. Pharmacological inhibition or knockout of nonmuscle myosin 2A prevents these adjustments, suggesting that cellular tensional homeostasis strongly depends on functional myosin motors.

Shaping the heart: Structural and functional maturation of iPSC-cardiomyocytes in 3D-micro-scaffolds.

Cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) represent the best cell source for cardiac regenerative purposes but retain an immature phenotype after differentiation with significant limitations compared to adult cardiomyocytes. Apart from an incomplete cardiomyocyte-specific structure and microarchitecture, cells show at the level of Ca2+ signaling only slow Ca2+ release and reuptake properties. Here, we investigated the effect of restructuring single iPSC-CMs in specially designed 3D-micro-scaffolds on cell morphology and Ca2+ handling. Using direct laser writing, rectangular-shaped scaffolds were produced and single iPSC-CMs were seeded into these forms. Structural analyses revealed strong sarcolemmal remodeling processes and myofilament reorientation in 3D-shaped cells leading to enhanced clustered expression of L-type Ca2+ channels and ryanodine receptors and consequently, to faster Ca2+ transient kinetics. Spontaneous beating activity was enhanced and Ca2+ handling was more robust compared to non-patterned cells. Overall, our data demonstrate for the first time significant improvement of Ca2+ signaling properties in reshaped iPSC-CMs indicative of functional maturation by structural remodeling.

Multi-Indicator Approach for Characterising Urban Green Space Provision at City and City-District Level in Germany.

This paper addresses the question of how a sustainable urban development can be supported through simple measured quantities in the context of the specific provision of green space and open space. The specific provision of green space is analysed based on a combination of six indicators that describe, on the one hand, the access of inhabitants to green spaces and on the other hand, the settlement character as well as the strong anthropogenic imprint on the urban landscape. The indicators were calculated and combined in a 9-cell matrix for classifying the areas studied. The implementation was carried out at two scales for all German cities with at least 50,000 inhabitants as well as exemplarily for city districts of eight big cities. The calculated indicator values for representing green characteristics decrease with increasing number of inhabitants, whereas the opposite relationship was obtained for the indicators of the grey characteristics. We show how the approach provides an in-depth morphological assessment of German cities ranking their scores from low to the high presence of grey/green characteristics.

Tension and Elasticity Contribute to Fibroblast Cell Shape in Three Dimensions.

The shape of animal cells is an important regulator for many essential processes such as cell migration or division. It is strongly determined by the organization of the actin cytoskeleton, which is also the main regulator of cell forces. Quantitative analysis of cell shape helps to reveal the physical processes underlying cell shape and forces, but it is notoriously difficult to conduct it in three dimensions. Here we use direct laser writing to create 3D open scaffolds for adhesion of connective tissue cells through well-defined adhesion platforms. Due to actomyosin contractility in the cell contour, characteristic invaginations lined by actin bundles form between adjacent adhesion sites. Using quantitative image processing and mathematical modeling, we demonstrate that the resulting shapes are determined not only by contractility, but also by elastic stress in the peripheral actin bundles. In this way, cells can generate higher forces than through contractility alone.

3D Laser Micro- and Nanoprinting: Challenges for Chemistry.

3D printing is a powerful emerging technology for the tailored fabrication of advanced functional materials. This Review summarizes the state-of-the art with regard to 3D laser micro- and nanoprinting and explores the chemical challenges limiting its full exploitation: from the development of advanced functional materials for applications in cell biology and electronics to the chemical barriers that need to be overcome to enable fast writing velocities with resolution below the diffraction limit. We further explore chemical means to enable direct laser writing of multiple materials in one resist by highly wavelength selective (λ-orthogonal) photochemical processes. Finally, chemical processes to construct adaptive 3D written structures that are able to respond to external stimuli, such as light, heat, pH value, or specific molecules, are highlighted, and advanced concepts for degradable scaffolds are explored.

Molecular Switch for Sub-Diffraction Laser Lithography by Photoenol Intermediate-State Cis-Trans Isomerization.

Recent developments in stimulated-emission depletion (STED) microscopy have led to a step change in the achievable resolution and allowed breaking the diffraction limit by large factors. The core principle is based on a reversible molecular switch, allowing for light-triggered activation and deactivation in combination with a laser focus that incorporates a point or line of zero intensity. In the past years, the concept has been transferred from microscopy to maskless laser lithography, namely direct laser writing (DLW), in order to overcome the diffraction limit for optical lithography. Herein, we propose and experimentally introduce a system that realizes such a molecular switch for lithography. Specifically, the population of intermediate-state photoenol isomers of α-methyl benzaldehydes generated by two-photon absorption at 700 nm fundamental wavelength can be reversibly depleted by simultaneous irradiation at 440 nm, suppressing the subsequent Diels-Alder cycloaddition reaction which constitutes the chemical core of the writing process. We demonstrate the potential of the proposed mechanism for STED-inspired DLW by covalently functionalizing the surface of glass substrates via the photoenol-driven STED-inspired process exploiting reversible photoenol activation with a polymerization initiator. Subsequently, macromolecules are grown from the functionalized areas and the spatially coded glass slides are characterized by atomic-force microscopy. Our approach allows lines with a full-width-at-half-maximum of down to 60 nm and line gratings with a lateral resolution of 100 nm to be written, both surpassing the diffraction limit.

IKKα controls ATG16L1 degradation to prevent ER stress during inflammation.

Inhibition of the IκB kinase complex (IKK) has been implicated in the therapy of several chronic inflammatory diseases including inflammatory bowel diseases. In this study, using mice with an inactivatable IKKα kinase (IkkαAA/AA), we show that loss of IKKα function markedly impairs epithelial regeneration in a model of acute colitis. Mechanistically, this is caused by compromised secretion of cytoprotective IL-18 from IKKα-mutant intestinal epithelial cells because of elevated caspase 12 activation during an enhanced unfolded protein response (UPR). Induction of the UPR is linked to decreased ATG16L1 stabilization in IkkαAA/AA mice. We demonstrate that both TNF-R and nucleotide-binding oligomerization domain stimulation promote ATG16L1 stabilization via IKKα-dependent phosphorylation of ATG16L1 at Ser278. Thus, we propose IKKα as a central mediator sensing both cytokine and microbial stimulation to suppress endoplasmic reticulum stress, thereby assuring antiinflammatory function during acute intestinal inflammation.

Photonic molecules with a tunable inter-cavity gap.

Optical micro-resonators have broad applications. They are used, for example, to enhance light-matter interactions in optical sensors or as model systems for investigating fundamental physical mechanisms in cavity quantum electrodynamics. Coupling two or more micro-cavities is particularly interesting as it enlarges the design freedom and the field of application. In this context, achieving tunability of the coupling strength and hence the inter-cavity gap is of utmost importance for adjusting the properties of the coupled micro-resonator system. In this paper, we report on a novel coupling approach that allows highly precise tuning of the coupling gap of polymeric micro-resonators that are fabricated side by side on a common substrate. We structure goblet-shaped whispering-gallery-mode resonators on an elastic silicone-based polymer substrate by direct laser writing. The silicone substrate is mechanically stretched in order to exploit the lateral shrinkage to reduce the coupling gap. Incorporating a laser dye into the micro-resonators transforms the cavities into micro-lasers that can be pumped optically. We have investigated the lasing emission by micro-photoluminescence spectroscopy, focusing on the spatial localization of the modes. Our results demonstrate the formation of photonic molecules consisting of two or even three resonators, for which the coupling strengths and hence the lasing performance can be precisely tuned. Flexibility and tunability are key elements in future photonics, making our approach interesting for various photonic applications. For instance, as our coupling approach can also be extended to larger cavity arrays, it might serve as a platform for tunable coupled-resonator optical waveguide devices.

Guiding Cell Attachment in 3D Microscaffolds Selectively Functionalized with Two Distinct Adhesion Proteins.

The combination of three different photoresists into a single direct laser written 3D microscaffold permits functionalization with two bioactive full-length proteins. The cell-instructive microscaffolds consist of a passivating framework equipped with light activatable constituents featuring distinct protein-binding properties. This allows directed cell attachment of epithelial or fibroblast cells in 3D.

Efficacy and tolerability of dimethyl fumarate in White-, African- and Hispanic- Americans with multiple sclerosis.

BACKGROUND: Dimethyl fumarate (DMF) was approved by the US Food and Drug Administration (FDA) for treatment of relapsing-remitting multiple sclerosis (RRMS) based on two phase III randomized clinical trials (RCTs). There were not enough non-White patients enrolled in these RCTs to allow for subgroup analysis based on race. Efficacy and tolerability of DMF therapy across various racial groups is unknown. METHODS: Retrospective chart review was performed on all patients with RRMS who were started on DMF in two tertiary multiple sclerosis (MS) clinics. Efficacy and tolerability of DMF was compared across three self-identified racial groups: White-American (WA), African-American (AA) and Hispanic-American (HA). RESULTS: A total of 390 RRMS patients were included in the study: 261 (66.9%) WA, 69 (17.7%) AA and 52 (13.3%) HA. When comparing 'pre-DMF' (1 year) and 'on DMF' (mean follow up of 14 months) periods, statistically significant reduction in rates of annualized relapses (WA from 0.44 to 0.19, AA from 0.39 to 0.15, and HA from 0.39 to 0.14; no differences between groups), new T2 lesions (WA from 45% to 23%, AA from 39% to 23%, HA from 52% to 26%; no difference between groups), and Gd+ lesions (WA from 25% to 13%, AA from 24% to 7%, HA from 23% to 12%; no difference between groups) were seen. DMF was relatively well tolerated across all groups, with an overall discontinuation rate of 20% (no difference between the three groups). CONCLUSION: Efficacy of DMF in our clinic population did not differ across three major ethnic groups, WA, AA and HA, and was comparable with results observed in the pivotal studies. These 'real-life' data suggest that race is not a factor that needs to be taken into account when initiating DMF.