As the power electronics landscape evolves, pushing for greater vertical integration, capillary underfilling is considered a versatile encapsulation technique suited for iterative development cycles of innovative integration concepts. Since a defect-free application is critical, this study proposes a capillary two-phase flow simulation, predicting both the flow pattern and velocity with remarkable precision and efficiency. In a preliminary performance evaluation, Volume of Fluid (VOF) outperforms the Level-Set method in terms of accuracy and computation time. Strategies like HRIC blending, artificial viscosity, and implicit Multi-Stepping prove effective in optimizing the numerical VOF scheme. Digital mapping using physical experiments and virtual simulations validates transient flow predictions, achieving excellent agreement with deviations as low as 1.48–3.34%. The accuracy of flow predictions is thereby greatly influenced by non-Newtonian viscosity characteristics in the low shear range and time-dependent contact angle variations. The study further explores flow manipulation concepts, focusing on local flow speed adjustment, gap segmentation, and the use of arcuate shapes to influence interface confluence near the chip. Experimental validation corroborates the effectiveness of each design intervention. In conclusion, this research highlights the potential of predictive engineering to develop flow-optimized package designs that enhance reliability while supporting high manufacturing yields. The integration of power electronics into compact modules has seen widespread adoption across various industries. As such, the advancement of electric vehicle inverters is driven by increasing requirements for power density and conversion efficiency, propelling a shift towards vertical integration as a viable alternative to conventional wire bonding approaches . TraditiIn response, the encapsulation of power modules assumes increasing importance to enhance dielectric, mechanical, and thermomechanical reliability. Present methodologies primarily entail transfer molding using package-specific molds with epoxy molding compounds (EMC). For this reason, recent advancements in compression molding technology have introduced a more flexible platform, enabling the encapsulation of diverse power packages via a single molding tool .This article focuses on capillary underfilling as a versatile encapsulation technique, particularly suited to the intricate iterative development phases of novel vertical integration concepts. Unlike traditional approaches, this method eliminates the need for custom molds and subsequent processing steps. Nonetheless, ensuring a defect-free underfill application presents a critical challenge, given that both reliability and production throughput are significantly impacted by void formation and prolonged filling times . Hence, In recent years, various studies have delved into the investigation of influential factors in underfill flow and the development of reliable models to predict capillary flow, albeit rarely in the field of power packaging . Ng and Aside from the numerical methods used, the main focus of these studies was the geometry of the classic flip-chip packaging with gap height, bump shape, and pitch being the most studied parameters concerning design optimization ,19,20,21This study applies the concept of a digital twin to the conventional underfilling of multi-chip power packages and presents a robust two-phase flow model as a basis for future package development. As an extended version, this paper covers two previous publications ,24. FirsThe following sections describe the materials used for physical experiments, material characterizations, and virtual mapping methods for experimental validation.2O3) and attached via solder joint (SnSb5) and a sintered Ag layer. Depending on the number of incorporated devices, the lateral dimensions can span up to 70 mm [This study primarily centers on the advancement of multi-chip half-bridge modules employing insulated gate bipolar transistors (IGBTs). These modules typically consist of pairs of 1200 V/600 A IGBTs along with a complementary free-wheeling diode, constituting a versatile multi-chip arrangement, as depicted in The proposed simulation method must therefore account for locally varying wetting properties and widely varying gap heights. Beyond the specific use case, the model can also be employed for smaller gap heights of e.g., 10–30 µm to describe the capillary liquid spreading of viscous, non-dispersive fluids in microchannels in general. This opens up further potential applications in electronics packaging such as FCBGAs , as wellTo verify the transient simulation result with experimental flow recordings, an innovative strategy was introduced. It is worth noting that replacing the actual package substrates with transparent material dummies ,29 chang2O3 substrates. For die imitation, a copper layer was attached via solder/sinter preforms. Substrate materials and dimensions are listed in To retain a representative material composition, the flow was monitored using thinned FR4 and AlTest samples for flow calibration without any overhangs within the gap were illuminated from underneath to guarantee high-contrast images;To include flow-optimizing design elements, dam underfill was applied before soldering. Benefits of this material include its limited wetting characteristics and its capacity to accommodate fluctuating gap dimensions;After setup, the gap height of each specimen was measured using confocal laser scanning microscopy. Considering substrate warping caused by thermal expansion during the joining process, average gap heights were computed with polynomial fitting;Translucency of the thinned substrates enabled real-time monitoring of the black-colored underfill, captured from a top view (xy-plane). Accordingly, this approach is well-suited for identifying voids across the entire gap height.The underfill was thawed to room temperature one hour prior to the experiments, while the assembly was preheated to 60 °C. Thermocouples were used to control the temperature ensuring a uniform surface temperature. The underfill was applied on the bottom substrate along the edges of the upper substrate using a variable dispensing pattern. Once the gap was filled, the curing process was initiated at 135 °C.The viscoelastic characteristics of the underfill material were examined through shear-controlled viscosity measurements at the process temperature of 60 °C. The measurements were performed with a gap size of 500 µm on a MCR 302 rheometer, Anton Paar GmbH. The measured viscosity values were interpolated for a direct use in the simulation model. In addition, the measured data were also fitted with the commonly used power law using a constant viscosity for shear rates lower than 10 1/s. Simulation results for both approaches a were coMoreover, time-dependent changes in viscosity were analyzed for isothermal conditions at 60 °C with a constant shear rate of 1/s, shown in 2O3, and Cu. Simulating the manufacturing process of considered packages, the samples were subjected to a sequential sinter and solder process prior to measurement. Each contact angle was analyzed over time to include wetting dynamics, as shown in Pendant drop and sessile drop methods at 60 °C  were adopted to measure the surface tension of the underfill and its contact angle on different solid surfaces, such as FR4, AlThe systematic evaluation of the deviations between numerical simulations and experimental results is illustrated in ® version 5.6 [This study evaluates the performance of an FEM-based approach using COMSOL Multiphysicssion 5.6  comparedsion 5.6 . Within Both Eulerian multiphase methods treat the two phases as a unified continuous mixture of immiscible fluids governed by the same velocity field. In this study, the focus is set on epoxy-based underfill materials with elevated filler content and small particle sizes. Assuming a homogenous material was thus seen as a justifiable simplification to minimize computational costs and to make the simulation applicable to subsequent iterative design efforts. Given minimal capillary pressure gradients and Reynolds numbers, a laminar flow is assumed. Changes in underfill viscosity during the filling process are considered b; howeveThe governing equations for both phases are represented by the continuity equation and the Navier–Stokes equation, which articulates the conservation of mass and momentum, respectively:The velocity and pressure fields  model . This inCapillary pressure is driven by the interplay of contact angle Previously, in classic flip-chip packaging (FCP), a common modeling strategy was to confine the computational domain to the gap volume between the chip and substrate. During the underfilling of FCBGAs, accelerated flow along the lateral chip edge was observed . This phThe model can be adjusted by introducing dynamic boundary conditions as functions of time and space. This is crucial for a dispensing path optimization when multiple dispensing positions are involved. It also becomes relevant if a single dispense line is extensive or the dispensing speed is low, resulting in an asymmetric flow front .The dominant material properties governing the flow are notably sensitive to temperature variations. In cases where the assembly experiences significant temperature gradients during the filling process, a coupled two-phase flow simulation with heat transfer becomes advisable. Nonetheless, experimentation involving thermocouples and preliminary simulations of steady-state heat transfer have demonstrated minimal temperature gradients along the gap height, due to uniform preheating. Thus, the benefit of coupling heat transfer over assuming isothermal flow conditions is marginal in this case.To evaluate the performance of the two interface-capturing methods Level-Set and Volume of Fluid, a mesh convergency study was performed. The Grid Convergence Index (GCI) was used as a quantifiable and standardized measure to gauge spatial discretization errors . A constThe filling time, which was defined as the time between initial dispensing and 90% coverage of the gap cavity, was calculated for a coarse fh=0Following the outcomes presented in Utilizing Comsol for further analysis would necessitate mesh refinement and therefore require even higher computational effort. The VOF model, in contrast, shows minimal discretization errors of 0.77% implying asymptotic convergence. Accordingly, VOF is considered superior to the Level-Set method in terms of efficiency and precision and is chosen for further analyses.The model faces considerable stability issues and extensive computation times due to limited capillary driving forces, extended filling durations, and pronounced interface discontinuities marked by significant variations in density and viscosity between underfill and air. This underscores the relevance of enhancing the chosen solver schemes to tailor the model to the specific use case requirements.For VOF modeling, this is mainly carried out using a parametric optimization of the High-Resolution Interface-Capturing (HRIC) method . The HRIϕ . BalanciWhile DDS yields favorable outcomes for situations featuring an interface perpendicular to the flow direction  model, was split into three equal substeps for the VOF solver. This allowed increasing the CFL limits for the Segregated Flow solver, resulting in a much larger global time step size, while still maintaining a precise interface resolution through smaller time steps for interface propagation calculations.As depicted in The computed filling time was selected as a target value to evaluate the impact of input parameter variations involving gap height, surface tension, contact angle, and viscosity within a range of In addition, modified analytical models, as that of Darbois Texier et al. , were deications ,53,54,55A simplified geometry was used to assess the flow velocity for varying gap heights  was introduced to slow down the flow by reducing the cross-sectional height. Vice versa, to intentionally accelerate the flow, the inclusion of non-functional metallization areas with easily wettable surfaces is an option. Note that any additional layer will reduce gap height, weakening the accelerating effect;Solder bump arrays may also be used for flow manipulation. Squared instead of cylindrical pillars  and smalHigher bump pitch for more manageable flow behavior;Stands as a reference flow without any modifications;Additional layers for lowering the contact angle are more efficient, if incorporated in a striped shape instead of full coverage. This can be beneficial when substrate depressions are not a viable option to increase the local gap height.In areas susceptible to voids, design elements can obstruct the flow to prevent unwanted confluences. Pockets, which do not allow for simultaneous inflow and outflow of the underfill (dead ends), can be split into curved channels. Dual-Chip module-inspired geometries, as illustrated in Drawing inspiration from biological vein valves, The previous chapters were intended to indicate the potential of using the simulation model as a design tool driving innovation to come up with novel ideas for flow-optimized designs. The derived concepts were finally tested for their effectiveness using the established validation approach.The results in In the context of encapsulating planar power packages, a numerical framework for capillary underfilling was successfully implemented, utilizing both Level-Set (Comsol) and Volume of Fluid (Star-CCM+) methods. Performance evaluation has favored VOF, providing more advanced capabilities for model calibration tailored to the demands of specific applications. Improvements in precision, stability, and computational efficiency were successfully achieved by optimizing the HRIC scheme. This involved making adjustments in discretization blending, incorporating additional dissipation terms, and implementing implicit Multi-Stepping. The tuned VOF model exhibits excellent accuracy in predicting flow patterns and flow velocities, validated through various experimental trials, showing deviations as minor as 1.48% to 3.34%. Moreover, geometries mimicking application-based designs were tested, showcasing the effectiveness of customized design features for flow manipulation.Backed by a robust simulation model, alongside effective validation strategies, this research provides a solid foundation for future studies in capillary underfilling design and process optimization. These findings hold significant implications for semiconductor packaging, advanced integrated circuits, and sensors, where void-free underfilling is critical for device reliability and performance. Beyond reactive troubleshooting, the simulation has evolved into a proactive and predictive tool that seamlessly integrates into the development workflow, encouraging early consideration of flow optimization strategies.On a broader scale, the proposed simulation method has versatile applications in modeling the wicking in microchannels between two plates, making it a valuable tool, for example, in microfluidics and lab-on-chip technologies relevant to healthcare, diagnostics, and chemical analysis.


Transverse myelitis is a rare but documented sequela of heroin use. While the underlying etiology is not clearly elucidated, the prevailing pathophysiologic mechanism amongst existing literature suggests an immune-mediated hypersensitivity reaction due to heroin insufflation following a long period of abstinence. Outcomes vary among the limited reports, but prognosis tends to be poor due to an acute and rapidly progressive disease course. Here, we describe a case of extensive transverse myelitis in a chronic heroin user following heroin insufflation. This report hopes to provide greater insight into the underlying cause of this rare phenomenon due to our patient’s discrepancy from the documented norm of heroin abstinence preceding disease onset. Transverse myelitis (TM) is a rare neurological condition characterized by inflammation and damage to the spinal cord with potentially severe long-term neurological morbidity and mortality. The etiology of TM varies, including a large spectrum of underlying causes including, but not limited to, drug/toxin, infectious, paraneoplastic, idiopathic, systemic autoimmune disorders, and acquired demyelinating diseases -2. QuickA 50-year-old man with a past medical history of heroin use and hypertension presented to the Emergency Department (ED) with acute onset right-sided focal weakness. The patient reported smoking heroin earlier that day and then napping for 4 hours. Shortly after waking, the patient noticed right-sided weakness with difficulty moving the right upper and lower extremities. The severity of the weakness became progressively worse, at which point the patient came to the ED. The symptom onset was roughly 5 hours after heroin insufflation. The patient denied any similar problems in the past and endorsed being a consistent heroin user. Past medical history was significant for consistent heroin use, hypertension, obesity, diabetes mellitus type 2, and hepatitis C. Vital signs were within normal limits. The initial physical exam showed weakness in the right upper and lower extremities with no sensory changes. Re-evaluation two hours after admission showed progressive weakness now spreading to the left extremities. Widespread loss of sensation and rapidly progressing paralysis soon occurred, ultimately requiring intubation due to decreased respiratory effort. Diagnosis of TM was confirmed with an MRI of the cervical spine revealing abnormal T2 signaling extending multiple levels down the central spinal cord tracts from C2-C7 with increased cord expansion suggestive of transverse myelitis Figure . This waNeurology was consulted for continued management. Our patient received a 5-day course of high-dose steroids with 1g IV methylprednisone. Unfortunately, his symptoms did not improve following high-dose steroids. During a neurological exam with the patient not on sedation, there was complete paralysis and loss of sensation in all extremities. The patient was able to blink and stick out his tongue in response to commands. Before intubation, the patient had a limited verbal response of grunting. The patient’s course was complicated by persistent rhabdomyolysis with elevated creatine kinase (CK) causing worsening renal failure, ultimately leading to the patient requiring hemodialysis. Eventually, a tracheostomy and percutaneous endoscopic gastrostomy tube were placed, given his continued ventilator dependency and inability to swallow. The patient was eventually discharged to a long-term acute care facility for extensive rehabilitation.To our knowledge, roughly 10 cases of heroin-induced TM are available in the scientific literature over approximately the last 50 years. Strong similarities have been noted between cases including most being secondary to heroin insufflation, disease onset following use after a period of abstinence, and the presence of rapid onset of symptoms often resulting in permanent neurological deficits and death. While high-dose steroids have been successful in halting progression in some patients, the prognosis is usually poor -9. A key difference in drug use history was present in our patient which may shed light on various theories as to why heroin-induced TM occurs. The greatest consistency amongst previous cases is heroin insufflation in a previous user following a period of non-use. This documented timeline strongly suggests an immune-mediated hypersensitivity reaction as being the underlying cause of heroin-induced TM. Ivanosvki et al clearly demonstrated this association by testing for opioid use through hair sampling to support a period of previous abstinence in their patient . ThroughMany different hypotheses have been proposed for the underlying mechanism behind heroin-induced TM, including vascular damage leading to ischemia, compression, direct/contaminant toxicity, and immunological effects from heroin or associated contaminants -8. WhileIschemia due to poor vascular supply has also been proposed as a possible mechanism ,7,10. CoOur report highlights the difficult diagnostic challenge of heroin-induced transverse myelitis and furthers our understanding of clinical features, diagnostics, treatment, and prognosis. As heroin-induced TM progresses rapidly, fast diagnosis and treatment are imperative. Obtaining a detailed patient history, thorough neurologic exam, and imaging is essential to diagnosis. Although there is limited efficacy, treatment with high-dose steroids is the recommended plan of action. Due to our case’s unique spontaneous onset of transverse myelitis during consistent heroin use, we would like to emphasize the impact this case report may have on the current understanding of the pathophysiology behind heroin-induced transverse myelitis as we compare autoimmune, vascular, and other etiologies.


Primary sclerosing cholangitis (PSC) is an idiopathic cholestatic liver disease with progressive inflammation of the intra- or extrahepatic bile ducts, leading to segmental fibrosis and, ultimately, cirrhosis. It is considered a rare disease, with a prevalence of approximately 10–16 cases per 100,000 individuals . Given tSeveral studies demonstrated an elevated risk of PSC among first-degree relatives . GWAS haVeillonella genus in patients with PSC-IBD when compared with both healthy controls and patients with IBD alone. To better evaluate a causative link between dysbiosis, intestinal permeability, and hepatobiliary effects, Nakamoto et al. transplanted fecal microbiota from patients with PSC into gnotobiotic mice and identified three bacterial strains  from their mesenteric lymph nodes  are tissue-resident immune cells that were observed to accumulate around the portal tracts in both human and animal models of PSC. Mice that were deficient in MCs exhibited decreased portal inflammation and ductular reaction, and these effects were linked to H2 histamine receptor signaling . MCs expAnother study showed that the FGF15/-19/FXR/CYP7A1 axis had a key role in modulating BA synthesis. In patients, suppressed BA synthesis portended a worse prognosis with low serum C4 levels . FXR agoTGR5 is a GPCR for primary and secondary BAs and was shown to have a protective role in biliary epithelial cells (BECs) by stimulating tight junction integrity. Reich et al. demonstrated TGR5 downregulation in PSC, and this contributed to a reactive BEC phenotype. Restoring TGR5 or increasing its level by norursodeoxycholic acid (norUDCA) resulted in improved liver enzymes and histology . Clinica–/–Mdr2 mice through miR-125b and FoxA2 , which is only expressed on cholangiocytes within the liver. Recently, long-term administration of the SR antagonist (SCT 5-27) decreased ductular reaction and liver fibrosis in bile duct–ligated and nd FoxA2 . Taken tBased on preclinical studies, FXR is an intriguing target for PSC studies. The results of the phase II and open-label extension trial of the FXR agonist cilofexor were promising, with improved liver enzymes and serum BA levels in noncirrhotic PSC patients . A largeDespite tremendous efforts to define the key pathogenic features and therapeutic targets, effective medical therapy for PSC is still lacking. There are several obstacles that may impede faster progress. PSC animal models do not faithfully phenocopy the complex nature of human disease, thus translation may not be straightforward. We are now beginning to understand the early triggering events, such as the role of microbiota and mucosal immunity and their effects on innate immunity in PSC, and there is a significant effort to develop new biomarkers with acceptable sensitivity and specificity. High-throughput methods could be useful for biomarker discovery, cellular landscape analysis, and identification of novel pathways and networks related to the disease. The combination of single-cell and high-resolution spatial transcriptomics could advance the field by identifying new subsets of immune cells and their connection to other cell types or the extracellular matrix.Furthermore, identification of the subsets of patients at high risk for disease progression or development of cholangiocarcinoma is critical. Improved surveillance involving a serum microRNA profile or AI-based technologies could help define predictive models .With all the recent discoveries, effective medical therapy is expected to be available in the next few years. This could involve using combination therapies, considering genetic susceptibilities, and targeting different disease pathways such as those for immune dysregulation, fibrosis, and dysbiosis, and manipulating the microbiome.


The ability to accurately map and manipulate the dynamic subcellular distribution of proteins is key for a mechanistic understanding of neuronal functioning. Current fluorescence microscopy techniques provide access to subcellular protein organization at increasing resolution but are often restricted by the availability of methods that reliably label endogenous proteins. Excitingly, recent development in CRISPR/Cas9 genome editing now allows researchers to specifically tag and visualize endogenous proteins, overcoming limitations associated with current labeling strategies. This article will discuss the progress that has been made in the last years that has led to the development of CRISPR/Cas9 genome editing tools for the reliable mapping of endogenous proteins in neurons. Furthermore, recently developed tools enable the duplex labeling of two proteins simultaneously and acute manipulation of protein distribution. Future implementations of this generation of genome editing technologies will undoubtedly drive progress in molecular and cellular neurobiology. Mapping the dynamic subcellular organization of proteins at high precision is therefore essential for our understanding of neuronal functioning.2––in vivo mouse brain.,,–,,,CRISPR/Cas9-based genome editing techniques allow the site-specific modification, removal, or addition of DNA segments. CRISPR/Cas9 relies on a guide RNA (gRNA) sequence containing a protospacer adjacent motif (PAM) that instructs Cas9 to induce a double-stranded DNA break (DSB) at a specified site in the genome. This DSB can trigger two endogenous DNA repair pathways: the non-homologous end-joining (NHEJ) pathway and the homology-directed repair (HDR) pathway. The HDR pathway can be utilized to direct a precise recombination event between a homologous donor sequence and the sequence around the DSB to generate site-specific knock-ins. HDR is however tightly controlled by the cell cycle and is strongly suppressed in post-mitotic cells, such as neurons. Nevertheless, HDR has been used successfully in neurons to generate knock-ins and visualize endogenously tagged proteins.3Multiplexing genome editing to tag two or more proteins simultaneously could allow for co-localization or interaction studies using for instance Förster resonance energy transfer. Multiplexing remained challenging however, because in NHEJ-based repair the donor sequence can be integrated in any DSB. Thus, when editing two loci simultaneously, the integration of specific donor sequences is untargeted, leading to crosstalk. One solution that was shown to generate double knock-ins,Reliable labeling of endogenous proteins in neurons is an important step, but for a mechanistic understanding of neuronal cell biology, tools are required that can precisely manipulate the abundance or localization of proteins. Targeted CRISPR/Cas9-mediated knock-out is relatively simple and effective and can be used to study protein function in neurons.4The exciting developments in the field of genome editing continue to drive progress in neuronal cell biology. Nevertheless, several improvements could be made to increase the utility of genome editing. First, the efficiency of genome editing in neurons is still relatively low. We found that the efficiency of individual knock-in vectors targeting the same gene, even targeting PAM sites spaced by only a few base pairs, could differ


Carcinosarcoma ex pleomorphic adenoma is a rare malignant neoplasm, with most cases reported in the parotid gland. We herein report a case of a 75-year-old male referred to our hospital with a long-standing right parotid lesion that was treated in an outside hospital by a superficial parotidectomy. The patient reported a painful, rapidly enlarging mass following the excision. Histopathological examination showed the proliferation of malignant epithelial and mesenchymal elements with a solid sheet of Langerhans cells admixed with eosinophils. The Langerhans cells were reactive to CD1a and BRAF; hence, a diagnosis of carcinosarcoma ex pleomorphic adenoma with Langerhans cell histiocytosis was given. Complete clinical and radiographic workup showed no other organ involvement. The patient underwent total parotidectomy with adjuvant chemoradiation; however, the tumor progressed and showed lung metastasis. We herein report the first case of a concurrent Langerhans cell histiocytosis with associated carcinosarcoma ex pleomorphic adenoma. Carcinosarcoma  comprises both malignant epithelial and malignant mesenchymal components. It is an exceedingly rare tumor of the salivary gland. Most cases arise de novo; however, some patients have a history of a long-standing or recurrent pleomorphic adenoma, a condition described as carcinosarcoma ex pleomorphic adenoma . It is uMicroscopically, the lesion shows a biphasic tumor component with the epithelial component showing multiple features, including squamous cell carcinoma (SCC), adenocarcinoma not otherwise specified (NOS), or salivary duct carcinoma (SDC). Less reported cases showed spindle cell carcinoma, mucoepidermoid carcinoma, intraductal carcinoma (IDC), and large cell neuroendocrine carcinoma (LCNEC). The mesenchymal component usually shows chondrosarcoma, osteosarcoma, fibrosarcoma, and rhabdomyosarcoma components. The less commonly reported mesenchymal components include undifferentiated pleomorphic sarcoma, liposarcoma, and myofibrosarcoma. Most cases showed a remnant of pleomorphic adenoma with a transition of the tumor from a benign component to an area that shows atypical pleomorphic adenoma to a frank malignant neoplasm. However, some cases show only fibromyxoid stroma or, more commonly, hyaline nodule as the benign component. Extensive necrosis is common with pleomorphism and increased mitosis -11. The Langerhans cell histiocytosis (LCH) is considered the most common histiocytic disease, characterized by the proliferation of langerin-positive histiocytes (CD207+) with infiltration of inflammatory cells. It can be a localized lesion or involve multiple organs, with the latter showing a more aggressive clinical course . LangerhA 75-year-old male was referred to our hospital for a recurrent right parotid mass. The patient reported that the lesion persisted for over 25 years and was slowly enlarging. After that, he received a superficial parotidectomy in an outside hospital. The diagnosis from the referring hospital was pleomorphic adenoma with extensive myxoid degeneration, and they mentioned that the tumor had an involved margin. The patient reported a rapidly enlarging painful mass and was referred to our hospital. The radiological findings showed a large heterogeneously enhancing lobulated solid cystic mass arising from the superficial lobe of the right parotid gland measuring around 10 cm with multiple solid enhancing nodules abutting the primary lesion  is considered the most common histiocytic disease, characterized by the proliferation of langerin-positive histiocytes (CD207+) with infiltration of inflammatory cells. It can be a localized lesion or involve multiple organs, with the latter showing a more aggressive clinical course . It coulCarcinosarcoma ex pleomorphic adenoma is usually managed surgically with adjuvant chemotherapy, adjuvant radiotherapy, or adjuvant chemoradiation; however, all treatment modalities yield a dismal prognosis with tumor progression or death from the disease, with rare cases reporting no tumor recurrence. However, these cases report no follow-up period or a minimum follow-up period -4,6-8. SWe herein report the first case report of a concurrent carcinosarcoma ex pleomorphic adenoma with Langerhans cell histiocytosis. Our case shows a long-standing parotid lesion that showed rapid enlargement after surgical intervention; unfortunately, the tumor progressed with the therapy.


In vivo MRI and an ex vivo clonogenic assay were used to determine pH effects on PAA-CONPs’ radioprotection in irradiated tumor-bearing mice. PAA-CONPs showed excellent radioprotective efficacy in the normal colon that was equivalent to uncoated CONPs and amifostine. [18F]FDG PET imaging showed PAA-CONPs do not affect tumor response to radiation. Normalization of tumor pH allowed some radioprotection of tumors by PAA-CONPs, which may explain their lack of tumor radioprotection in the acidic tumor microenvironment. Overall, PAA-CONPs meet the criteria for clinical application as a radioprotective therapeutic agent and are an excellent candidate for further study.Cerium oxide nanoparticles (CONPs) have a unique surface redox chemistry that appears to selectively protect normal tissues from radiation induced damage. Our prior research exploring the biocompatibility of polymer-coated CONPs found further study of poly-acrylic acid (PAA)-coated CONPs was warranted due to improved systemic biodistribution and rapid renal clearance. This work further explores PAA-CONPs’ radioprotective efficacy and mechanism of action related to tumor microenvironment pH. An ex vivo TUNEL assay was used to measure PAA-CONPs’ protection of the irradiated mouse colon in comparison to the established radioprotector amifostine. [ Despite advances in cancer treatment using radiation therapy (RT), this modality of treatment is inherently limited by damage to normal tissues surrounding the targeted cancer. Side effects from normal tissue damage can drastically decrease the quality of life of patients undergoing therapy ,2,3,4. TThe use of radioprotectors in the clinic has been limited due to the limited number of compounds having the required properties for appropriate use in humans . Firstly3+ and Ce4+, with oxygen vacancies, which allows them to mimic the catalytic activity of enzymes such as superoxide dismutase (SOD), catalase, oxidases, and peroxidases FDG PET imaging, the colons were irradiated on the SARRP. Two weeks after irradiation, [18F]FDG PET imaging was repeated to determine tumor response.To evaluate the effects of CONPs on tumor response to radiation, [18F]FDG PET images was completed in Inveon Research Workplace 4.1 . Regions of interest (ROIs) were created in the coronal plane outlining the colon tumors, careful to avoid inclusion of the bladder due to the high urine content of [18F]FDG. Tumor volumes were determined using a threshold standardized uptake value (SUV) of 2.5 for all images, which corrects for the injected dose and body weight of the animal and excludes surrounding [18F]FDG tissue uptake. Analysis of FDG PET imaging of untreated control mice, irradiated mice, and mice pre-treated with PAA-CONP and uncoated CONP prior to irradiation. p < 0.05). Tumor volume changes were not statistically significant between irradiated mice versus either of the CONP treatments, though uncoated CONPs showed a trend toward poorer tumor control .Changes in the volume of spontaneous colon tumors over the course of 2 weeks were calculated based on FDG PET imaging of spontaneous colon tumors before and after irradiation with PAA-CONPs and uncoated CONPs pre-treatment showed that both did not prevent tumor regression after irradiation. However, pre-treatment with uncoated CONPs resulted in less tumor regression than pre-treatment with PAA-CONPs or irradiation alone. While not statistically significant, this trend may indicate some radioprotection by uncoated CONPs, which may impact tumor control and clinical outcomes. PAA-CONP treatment, however, showed the same level of tumor regression compared to irradiation alone, indicating it showed no protection of the tumor. This property makes them ideal for clinical application to ensure their action as a radioprotective drug does not adversely affect tumor control during radiation therapy.FDG PET data from irradiated spontaneous colon tumors may indicate this property is only present in certain tumor types or under certain tumor microenvironment conditions. A study of CONPs anchored to graphdiyne and used in cell culture and xenograft tumors has suggested that O2 production from CONPs’ catalase-mimic activity may alleviate hypoxia and increase radiation effectiveness in tumors FDG PET and MR imaging and an ex vivo clonogenic assay. This lack of radioprotection in tumors seems to be related to their reduced redox potential when exposed to the lower pH of the tumor microenvironment. Building on prior research, the results of this study demonstrate that PAA-CONPs appear to meet all the criteria of an ideal radioprotective therapeutic agent. While further toxicity studies are warranted, prior research has shown CONPs have a limited toxicity profile, and the dose of CONPs used in this study was well tolerated with no clinically evident adverse effects seen in treated mice. Further dose-response investigation is also warranted, but our study shows that CONPs exhibited their radioprotective effects at a dose much less than the dose of amifostine used, which was chosen to be comparable to the therapeutic dose in humans. With their low-dose efficacy, excellent biodistribution, and favorable pharmacokinetics with rapid renal clearance, the overall toxicity of PAA-CONPs may be much less than that seen with amifostine while affording the same radioprotective effects and not interfering with tumor control during radiation therapy. Overall, PAA-CONPs appear to be excellent radioprotective therapeutic candidates worthy of future study. This research suggests that PAA-CONPs have similar radioprotective efficacy as the established radioprotective therapeutic agent amifostine by showing equivalent protection of normal mouse colon tissue. This work also demonstrates that PAA-CONPs do not show protection of tumor tissue in both spontaneous and xenograft colon tumor models using in vivo [


Oral semaglutide use plus background treatment resulted in better outcomes than background treatment alone or sitagliptin plus background treatment, and similar outcomes to liraglutide plus background treatment or empagliflozin plus background treatment with overlapping 95% confidence ranges for QALYs. Oral semaglutide was estimated to be cost-effective compared with liraglutide plus background treatment and to have incremental cost-effectiveness ratios (ICERs) between $100,000 and $150,000 per QALY vs sitagliptin plus background treatment and background therapy alone, but was unlikely to meet these thresholds compared with empagliflozin plus background treatment.We agree with Kohli-Lynch et al that extended dominance is an appropriate approach when evaluating the full landscape of treatment options to identify the most cost-effective option within a class of drugs and/or for a particular indication. We also agree that empagliflozin would be the preferred add-on treatment in this analysis, since it gains comparable (though lower on average) QALYs with oral semaglutide at a lower  lifetime cost.However, this was not the aim of our analysis. We used pairwise comparisons of oral semaglutide vs each chosen comparator instead of calculating ICERs relative to the next least costly option because our overall goal was to separately evaluate the novel agent, oral semaglutide, compared with the 4 existing comparators. We did not perform an overall cross-class evaluation so as to determine the ordering of treatment value; rather, we sought to inform decision makers as they consider the place of oral semaglutide in the current treatment landscape.Regarding the threshold cost, we thank Kohli-Lynch et al for bringing this to our attention. The calculated oral semaglutide price needed to reach a $100,000 willingness-to-pay per QALY threshold was due to (a) the fact that empagliflozin had lower QALYs compared with oral semaglutide in 73% of simulations and (b) the microsimulation modeling approach used to calculate each price threshold. Each threshold value was back-calculated from individual runs  of individual patient-level simulations per comparison. In the case of oral semaglutide vs empagliflozin, the 95% credible range for incremental QALYs spans zero, with 27% of the simulations having lower QALYs for oral semaglutide, whereas oral semaglutide had fully positive incremental QALY credible ranges vs the other comparators. Therefore, cost-effectiveness plane quadrant crossing unfortunately makes a threshold price difficult to simply calculate from the overall costs and outcomes of the 2 treatments.


The development and bottom-up assembly of synthetic cells with a functional cytoskeleton sets a major milestone to understand cell mechanics and to develop man-made machines on the nano- and microscale. However, natural cytoskeletal components can be difficult to purify, deliberately engineer and reconstitute within synthetic cells which therefore limits the realization of multifaceted functions of modern cytoskeletons in synthetic cells. Here, we review recent progress in the development of synthetic cytoskeletons made from deoxyribonucleic acid (DNA) as a complementary strategy. In particular, we explore the capabilities and limitations of DNA cytoskeletons to mimic functions of natural cystoskeletons like reversible assembly, cargo transport, force generation, mechanical support and guided polymerization. With recent examples, we showcase the power of rationally designed DNA cytoskeletons for bottom-up assembled synthetic cells as fully engineerable entities. Nevertheless, the realization of dynamic instability, self-replication and genetic encoding as well as contractile force generating motors remains a fruitful challenge for the complete integration of multifunctional DNA-based cytoskeletons into synthetic cells. An essential component of every modern eukaryotic cell is the cytoskeleton because it drives cell motility, stability and intracellular transport, among other functions . In thisDNA nanotechnology allows one to programmably assemble DNA strands into higher-order structures using the complementary base pairing rules. These structures can be classified into several basic building blocks, including DNA linkers, Y-motifs, DNA tiles and DNA origami, which again can be assembled into larger architectures like DNA lattices and DNA nanotubes in a hierarchical manner . The potThe challenge towards the assembly of a DNA-based cytoskeleton is that it should not just perform one function but execute several different tasks on demand. DNA nanotubes are an important early type of DNA nanostructures  and visu. 2The key challenge for building DNA cytoskeletons is to reach the same sophistication as natural cytoskeletal elements using DNA as an entirely different building material with distinct chemical and physical properties. As a starting point, we first explore already existing DNA nanostructures that mimic one specific key property of natural cytoskeletons. Then, we discuss how they could be merged into one multifunctional synthetic cytoskeleton for a synthetic cell and which elements are still missing, providing exciting directions for future research.. 2.1Natural cells are mechanically stable in order to resist stress from the environment. However, their mechanical properties strongly depend on the cell type, i.e. a red blood cell is more deformable than an epithelial cell . This goa). A different approach is the targeted functionalization of individual DNA strands with hydrophobic moieties that incorporate in the lipid bilayer of GUVs  or  or d). I. I−1, whse H (b)  or trans (b) [c) . While tanotubes . By modiubes [d) . Moreoveanotubes e the carAll in all, these versatile strategies to induce cargo transport along DNA nanostructures make up a large toolbox for further studies within the confinement of GUVs.. 2.5a) [1F0-ATP synthase DNA origami levers have been attached to a mulitlayer DNA origami base [figure 6b) [figure 6c) [Cytoskeletal filaments can exert forces within natural cells or even cell communities based on their contractility in the presence of motor proteins like myosin II. The quest for DNA nanomachines that can exert similar forces on DNA nanostructures has resulted in quite a few DNA-based applications towards this aim ,76. By ffigure 6a) ,80. Simia) –84. Intea) . Towardsami base –87. Withgure 6b) . Moreovegure 6b) . A mechagure 6c) . Anothergure 6c) . In this. 3The development of more and more complex synthetic cells from the bottom up has deepened our understanding of natural cells. A major focus has been the engineering and reconstitution of natural cytoskeletons into cell-sized compartments. Recent advances of DNA nanotechnology in designing programmable micrometre-sized structures that can be interfaced with GUVs open up a new path towards entirely synthetic cytoskeletons for synthetic cells, engineered from nucleic acids rather than proteins. Our continuously increasing understanding of DNA as a building block adds to the comparably easy tuneability of biophysically relevant parameters with DNA. These include size, shape, diameter, charge, functionalization, persistence and rigidity, which can be easily engineered and altered with DNA nanotechnology. Additionally, the use of DNA nanotechnology is straightforward by making use of the large toolbox of versatile structures and corresponding libraries .These advancements have led to different solutions to establish the versatile functions of cytoskeletons with DNA like morphology control, reversible assembly, symmetry breaking, intracellular transport and force generation. As next challenges we identify (i) the incorporation of all these functionalities into a single DNA nanostructure, (ii) the incorporation of these multifunctional cytoskeletons into GUVs, (iii) mechanisms that allow the micrometre-sized contraction of DNA nanostructures and (iv) catalytic activity and self-regeneration.Even though a key advantage of DNA nanotechnology is the amount of different nanostructures that perform different functions , cytoskeThe next crucial step towards synthetic cytoskeletons for synthetic cells is the implementation of these into GUVs. Whereas all of the discussed cytoskeletal functions have been achieved in bulk, only some have been realized inside the confinement of lipid vesicles . TherefoAfter having established the reconstitution of multifunctional synthetic DNA cytoskeletons into GUVs, it will be essential to improve the current strength of forces and transport velocities that can be generated by DNA cytoskeletons. As cytoskeletal forces drive cell motility by balancing filament polymerization and contraction it will be an important step for the future success of DNA cytoskeletons to achieve the active stimuli-responsive remodelling of the GUV membrane from the inside. Moreover, this mechanism can be directly related to the division of GUVs as synthetic cell compartments, e.g. by engineering DNA rings that can contract and induce vesicle fission.All in all, the development of synthetic DNA-based cytoskeletons for synthetic cells with unique multifunctionality and straightforward tuneability will be an exciting step in the bottom-up assembly of truly synthetic cells from molecular building blocks. Thereby, DNA cytoskeletons will not only advance our knowledge in DNA nanotechnology but also serve as well-characterized model systems to understand natural cytoskeletons. Along this way, they allow the assembly of rationally engineered complex synthetic cells with applications in biomedicine, cellular biophysics and synthetic biology.


Current resin composites have favorable handling and upon polymerization initial physical properties that allow for efficient material replacement of removed carious tooth structure. Dental resin composites have long term durability limitations due to the hydrolysis of ester bonds within the methacrylate based polymer matrix. This article outlines the importance of ester bonds positioned internal to the carbon-carbon double bond in current methacrylate monomers. Water and promiscuous salivary/bacterial esterase activity can initiate ester bond hydrolysis that can sever the polymer backbone throughout the material. Recent studies have custom synthesized, with the latest advances in modern organic chemical synthesis, a novel molecule named ethylene glycol bis (ethyl methacrylate) (EGEMA). EGEMA was designed to retain the reactive acrylate units. Upon intermolecular polymerization of vinyl groups, EGEMA ester groups are positioned outside the backbone of the polymer chain. This review highlights investigation into the degradation resistance of EGEMA using buffer, esterase, and microbial storage assays. Material samples of EGEMA had superior final physical and mechanical properties than traditional ethylene glycol dimethacrylate (EGDMA) in all degradation assays. Integrating bioinformatics-based biodegradation predictions to the experimental results of storage media analyzed by LC/GC-MS revealed that hydrolysis of EGEMA generated small amounts of ethanol while preserving the strength bearing polymer backbone. Prior studies support investigation into additional custom synthesized methacrylate polymers with “flipped external” ester groups. The long term goal is to improve clinical durability compared to current methacrylates while retaining inherent advantages of acrylic based chemistry, which may ease implementation of these novel methacrylates into clinical practice. The majority of current resin based composite restorations use methacrylates as a polymer matrix. Despite clinical improvements compared to older generations, current methacrylate-based materials have less than ideal long term durability, especially in specific high risk populations –5. For hLaboratory assessment of current methacrylate materials have identified several weaknesses that affect long term stability of current composite restorations –12. PoteAn obvious solution to addressing methacrylate ester bond degradation is using a replacement chemistry that shares many of the short term advantages while improving upon the long-term durability concern. Ether-based polymers have notable potential as being resistant to hydrolytic degradation . Vinyl sAnother potential solution is re-designing the methacrylate polymer linkage system. For current methacrylates, the ester bond hydrolysis that severs the internal polymer backbone leads to a reduction in bulk mechanical and physical properties. A new approach is to design monomers that when they polymerize lack ester, carbonate, acetal, and anhydride bonds within their polymeric backbone sequence.In this brief review, recent research on repositioning or “flipping” the ester bond to an external position will be summarized. The aim of the review is to highlight the current evidence on the potential benefits of methacrylate monomers with flipped external ester groups. This review will also introduce the flipped methacrylate design as a polymer that has potential use in high-caries-risk populations such as pediatric patients where there is a need to not only improve upon outcomes for resin composite restorations, but also to increase durability for preventive sealants.EGDMA is used ubiquitously in dental materials. It is a hydrophilic monomer that is used as a diluent and mixed with larger strength-bearing hydrophobic monomers. EGDMA is commercially available and has been the starting point molecule for investigation into the influence of ester bond positioning. Current methacrylate monomers such as EGDMA can polymerize via a free radical mechanism mediated by a photoinitiator/co-initiators system of camphorquinone (CQ), ethyl 4-dimethylamino benzoate (EDMAB), and diphenyliodonium hexafluorophosphate (DPIHP). CQ/EDMAB/DPIHP combination has been found to be a highly efficient photoinitiator system and is activated by high-intensity light with a wavelength range of 385–515 nm . EGDMA cin situ with two equivalents of the electrophile ethyl 2-bromomethylacrylate to generate EGEMA (Recent studies have custom synthesized a novel molecule named ethylene glycol bis (ethyl methacrylate) (EGEMA) with the latest advances in modern organic chemical synthesis , 27. Unlte EGEMA  28)..in situ The main difference between EGEMA and EGDMA is the position of the ester groups in the monomer ,A. This 2) light (Recent studies demonstrated that EGEMA and EGDMA can photo-polymerized with similar degrees of conversion after 40 second exposure to 385–515 nm high intensity (1000 mW/cm2) light , 30, 31.2) light . These aWhile EGDMA demonstrated initial higher mechanical properties after curing compared to EGEMA, this characterization did not predict the long term durability trend of the two materials. Degradation assays examined EGDMA and EGEMA under various aqueous storage conditions. Materials were stored in solutions with increasing hydrolytic challenge to accelerate aging and investigate the hypothesis that ester bond positioning was a primary factor in the loss of mechanical strength. In order to interpret EGEMA and EGDMA results from these various degradation assays, it is essential to review the rationale for choosing specific degradation assays used in previous studies.While there are numerous storage solutions that tests dental material degradation potential using artificial aging models, phosphate buffered saline buffer (1xPBS) solutions at the resting saliva pH of 7.4 offers a starting point to understand hydrolysis of ester bonds. Materials can be stored at room temperature or at elevated temperatures. The advantage of elevated temperature storage is that higher temperature can accelerate material aging by increasing the rate of hydrolysis to shorten the study time . MateriaPseudomonas sp. (EC 3.1.1.13) at 0.5 units/ml concentration in 1x PBS can be used. This CEase has reproducible esterase activity that is measurable with a colorimetric p-nitrophenol acetate assay  provides a measure of durability to an acidogenic bacterium. S. mutans can potentially degrade material discs via enzyme and acid formation. S. mutans produces a known esterase with measurably activity , mechanical and physical properties changes occurred for both materials  were creating surface softening though ester bond hydrolysis. It is not surprising that surface properties showed differences between the temperature accelerated aging and CEase/S. mutans assays.In the three degradation studies, hardness values were less intuitive to interpret, since there were several factors happening during polymer disc storage. EGEMA and EGDMA both underwent a dark/post cure while in the storage solutions. In these degradation studies, hardness was defined as “selective intact surface hardness” since after aqueous storage hardness measurement needed to be measured on intact smooth surfaces. The testing instrumentation (Micro surface Vickers Hardness Test) was not capable of measuring hardness on irregular surface roughness caused by hydrolysis degradation. Nonetheless, the hardness results were interpreted in the context that there were two competing factors affecting surface hardness outside of the restraint of choosing intact smooth surfaces to measure. One factor was that the surface was getting harder through a dark/post cure, and the other factor was that the storage solutions /mass spectroscopy (MS) .Elucidating the unique biodegradation pathways of EGEMA and EGDMA was accomplished by integrating bioinformatics-based biodegradation predictions to the experimental results of storage media analyzed by LC/GC-MS. After samples were exposed for 15 weeks in PBS buffer solution and 9 weeks of CEase, storage solution aliquots were analyzed with chromatographic separation followed by in-line mass spectrometry. The molecular structures of EGDMA and EGEMA were also queried via the EAWAG-Biocatalysis/Biodegradation (BB) Prediction Pathway System (PPS) . The bioLC/MS analysis of EGDMA and EGEMA polymer discs measured 9 and 12 unique degradation by-products respectively. LC/MS analysis of EGEMA included the very likely byproducts of 2-hydroxyethyl 2-methylprop-2-enoate (HEMA) and methacrylic acid . LC/MS aThe low molecular weight by-products of ethylene glycol, in the case of the EGDMA pathway, and ethanol, in the case of EGEMA were not identified with LC-MS. It required GC-MS analysis to identify the very likely degradation by-products of ethylene glycol and ethanol that degraded from the two polymer discs. Ethanol was found in both 37°C buffer and CEase assay storage solutions, whereas ethylene glycol was found in greater frequency in the CEase assay which might be explained by the high rate of degradation in that assay compared with 15 week buffer storage.LC/GC-MS, integrated with a bioinformatics PPS, demonstrated that polymer backbone degradation occurred during the degradation of EGDMA, whereas EGEMA, with the external or flipped position of ester groups to the polymer backbone, had severing of side chains with the polymer backbone intact. The different ester bond positioning changed the chemical degradation pathway, by-products, and durability of the polymer discs.Altering the ester-bond linkage retains the benefits of methacrylate chemistry and the ease of commercial implementation while possibly increasing clinical longevity of resin composite restorations. Near-term investigation into substituting EGEMA for EGDMA is immediately achievable since EGEMA can be polymerized with the same photoinitiator system. Investigating EGEMA vs. EGDMA is a starting point for synthesis of many different flipped ester design methacrylate based on currently used methacrylates.In terms of immediate improvement, the diluent EGDMA is quite hydrophilic and is likely a weak link within the polymer mixture of a resin composite system. The hydrophilicity enhances water sorption that can lead to ester bond hydrolysis. Since the synthesis reaction is worked out for EGEMA, near future work can examine EGEMA substituting for EGDMA in an EGEMA/Bis-GMA resin mixture. This EGEMA/Bis-GMA mixture can be compared with EGDMA/Bis-GMA mixture. Future work is needed to investigate subsurface degradation from water and also investigate if CEase penetration into EDGMA/EGEMA discs occurs through microcracks caused from polymerization. The degradation effects of CEase may be reduced when EGDMA and EGEMA are co-polymerized with strength bearing Bis-GMA or a flipped ester design analog of Bis-GMA.Dimethyl diphenylmethane ethyl methacrylate (DMDPEMA) is flipped ester design analog of Bis-GMA that can be potentially blended with EGEMA. To achieve desirable handling properties, the addition of the low viscosity diluent EGEMA will be necessary since DMDPEMA has higher viscosity. The viscosity of DMDPEMA was not quantitatively measured yet, but it is qualitatively more viscous than EGEMA, and it is likely in the same order of magnitude as Bis-GMA. Bis-GMA needs to be blended with low viscosity EGDMA, and future work can compare DMDPEMA/EGEMA with Bis-GMA/EGDMA blends. Future work is needed to investigate DMDPEMA degradation compared to Bis-GMA degradation. Bis-GMA degradation involves breakage of the polymeric backbone yielding a main by-product of bis-hydroxy-propoxy-phenyl propane (Bis-HPPP) and methacrylic acid  43). Wh. Wh43). The future direction of flipped ester group design polymers first requires investigation into custom synthesis of these molecules. This will be a lengthy and ongoing area of investigation. After synthesizing and purifying these flipped ester groups, investigation needs to examine how the flipped design may in some cases create steric hindrances. There may be some polymer designs that are more favorable. Future examinations will address adding branch designs that may improve shrinkage, and based on traditional ester linkage methacrylates, branching can be designed to have comparable polymerization as linear monomers . Importain vitro cell studies, immunological based in vivo animal studies should be performed prior to human clinical trials. More work is needed to understand the biological properties and effects of methacrylate polymers with “flipped external” ester groups prior to clinical testing. For initial clinical testing, there may be benefits of optimizing dental sealants in pediatric populations as an initial clinical step. Dental sealants can be simply applied to acid etched dental enamel. An EGEMA/DMDPEMA blend is expected to be transparent to near infrared (1310-nm) light and optical coherence tomography may be utilized to assess initial sealant adaptation and longitudinal assessment of marginal integrity  can degrade the dentin-collagen hybrid layer, dentin bonding agents contain the same internal positioned ester bonds that can degrade EGDMA and other composites. The degradation effects of dentin adhesive hydrolysis is substantial. Even with MMP inhibitor pretreatment, water storage reduces microtensile bond strength of current adhesives and increases the marginal leakage 41% vs. initial properties . CohesivThe trajectory of research into flipped external ester group methacrylates can test these materials in high failure risk pediatric and adult dental patients. The traditional methacrylate design was optimized in a dental healthcare system that is currently changing. Clinical outcomes, rather than application time alone, are becoming an increasingly important metric for the dental profession and insurers . The cos


According to the literature, individuals who start tobacco smoking during adolescence are at greater risk of developing severe tobacco addiction and heavier smoking behavior in comparison with individuals who uptake tobacco smoking during subsequent developmental stages. As suggested by animal models, this may be related to the unique neuroadaptive and neurotoxic effects of nicotine on adolescents' fronto-striatal brain regions modulating cognitive control and impulsivity. Previous research has proposed that these neuroadaptive and neurotoxic effects may cause a heightened reward-oriented impulsive behavior that may foster smoking relapses during quit attempts. However, developments in the field of addiction neuroscience have proposed drug addiction to represent a type of compulsive behavior characterized by the persistent use of a particular drug despite evident adverse consequences. One brain region that has received increased attention in recent years and that has been proposed to play a central role in modulating such compulsive drug-seeking and using behavior is the insular cortex. Lesion studies have shown that structural damages in the insular cortex may disrupt smoking behavior, while neuroimaging studies reported lower gray matter volume in the anterior insular cortex of chronic smokers compared with non-smokers, in addition to correlations between gray matter volume in the anterior insular cortex and measures of compulsive cigarette smoking. Based on the findings of our recent study reporting on early-onset smokers (mean age at regular smoking initiation = 13.2 years) displaying lower gray matter and white matter volume in the anterior insular cortex compared to late-onset smokers (mean age at regular smoking initiation = 18.0 years), we propose that the anterior insular cortex may play a central role in mediating the association between smoking uptake during adolescence and smoking heaviness/tobacco addiction during adulthood. Tobacco smoking is the leading cause of preventable deaths worldwide. According to the WHO, approximately 8 million individuals die each year as the direct and indirect consequences of chronic tobacco smoking . TobaccoIndividuals with tobacco addiction usually start this maladaptive behavior during adolescence (before 21 years of age) –5. A genIn line with the above evidence, Debry and Tiffany  proposedHowever, in the last decade, several scholars in the field of addiction neuroscience have proposed drug addiction (including tobacco addiction) to represent a type of compulsive behavior that is characterized by a tendency toward repetitive, habitual actions, repeated despite adverse consequences . For insAccording to Figee et al. , the comeach subregion has different cytoarchitectonic features, connectivity, and therefore functions”. Particularly, functional connectivity studies have shown the posterior insula to be connected with brain regions modulating somatosensory and motor processing including parietal, occipital, and temporal cortices , and neuroimaging studies have shown that the anterior insula modulates the switching from the internally oriented default mode network (DMN) to the externally oriented executive control network (CEN)  is a brain region that has received increased attention in the last decades and has been increasingly recognized as pivotal in the development of compulsive drug use in the field of addiction neuroscience, albeit its mechanisms are still largely unknown. From a neuroanatomical point of view, the insular cortex can be roughly divided into anterior and posterior insula subregions. As stated by Namkung et al. , “each scortices , 18, andcortices . A recencortices  proposedrk (CEN) . The rigetworks” . Indeed,etworks” .Therefore, it is plausible to think that functional and structural disruptions of the anterior insular cortex (particularly of the right anterior insular cortex) may impair the interoceptive awareness of individuals with and their capacity to switch their attention toward more adaptive external stimuli; that is, they may continue to engage in compulsive drug taking as they may be less aware of the negative physiological effects of a particular drug [i.e. self-administration in the face of punishment or aversive consequences as proposed by Figee et al. ]. An fMRThe CDS assesses an individual's subjective experience of symptoms such as craving, compulsion to use, levels of stress when unable to smoke, and difficulty quitting or controlling intake”  and compulsive water intake  in 140 rats that underwent a neurosurgical and neuroimaging procedure . By also considering the strong association between smoking initiation during adolescence and severe tobacco addiction during adulthood –6, we raRecently, we conducted a VBM study hypothesizing that 11 adult smokers (mean age at recruitment = 25.2 years) who started regular tobacco use at 13.2 years of age  would present heightened impulsive and risky choices, in addition to reduced GM and WM volume in frontal brain regions modulating such behaviors in comparison with 17 adult smokers (mean age at recruitment= 30 years) who started regular smoking at 18 years of age  and 24 matched non-smoker controls . Our hypFrom a neuroanatomical point of view, early-onset smokers displayed lower GM volume in the right ACC and lower WM volume in the left anterior corpus callosum, left anterior insular cortex, and bilateral thalamus in comparison with non-smokers . RemarkaIt is worth noticing that early-onset smokers and late-onset tobacco smokers were well-matched in relation to tobacco use/exposure characteristics . Early-onset tobacco smokers displayed higher scores on the FTND in comparison with late-onset smokers albeit this difference did not reach statistical significance. No correlations were identified between tobacco use/exposure characteristics and GM/WM volume of early-onset smokers' anterior insular cortex. As reported in our previous study, this may indicate that such structural abnormalities were not due to differences in tobacco exposure between the two groups .Considering the findings from the above study, in addition to the evidence pertaining to the pivotal role of the anterior insular cortex in mediating tobacco addiction and compulsive drug use, we have developed a theoretical model with the aim to motivate future research investigating the relationship between smoking initiation during adolescence and tobacco addiction/compulsive smoking during adulthood under a neurocognitive point of view .The postulation of the above model  encompasMechanistically, the transition from an impulsive “reward-oriented” smoking behavior to compulsive smoking may be facilitated by the neurostructural and neurochemical alterations caused by the chronic consumption of tobacco on relevant brain regions  that are still developing during adolescence. Indeed, a neuroimaging study conducted by Dennis et al.  showed taddiction may reflect a discrepancy between an individual's predicted vs. actual internal state known as the bodily prediction error, an imbalance that could in turn adversely influence the degree of future drug-related approach vs. avoidance behavior”  interoceptive stimuli during risk-processing tasks –45. Indeehavior” . This maehavior” , 20. NotAccording to Robbins and Everitt , the losPoor response inhibition could also be considered a neurocognitive facet of this hypothesized compulsive smoking behavior as impairments in response inhibition are a predominant feature of psychiatric disorders characterized by high levels of compulsivity  . Indeed,could actually reflect a more sensitized (or overactive) interoceptive insular cortex system”  or overactivation to smoking cues (and its possible role in modulating tobacco cravings). The sample recruited in our VBM study was also small  and may These pathways are dissociable but they are neither mutually exclusive nor exhaustive. They may operate dynamically within the same individual at different times”  is not a unitary construct and may encompass three dissociable cognitive, motivational, and behavioral pathways . For inst times” . Other st times” .Nonetheless, considering the strong association between the structural and functional abnormalities in the anterior insular cortex and what we have defined as a “compulsive” tobacco smoking behavior  and by also considering the correlation between early-onset smoking and heaviness of tobacco smoking during adulthood –6, futurReplicating our structural neuroimaging findings by recruiting a larger sample size and by utilizing neurocognitive tasks assessing the multifaceted nature of compulsivity , response inhibition, interoceptive awareness, and the compulsive smoking behavior of participants by utilizing measures such as the CDS. These studies should also investigate possible differences in interoceptive awareness between early-onset smokers and late-onset smokers and may correlate GM/WM volume in the anterior insular cortex of early-onset smokers with measures of interoceptive awareness, scores of neurocognitive tasks, and measures of compulsive smoking behavior.Conducting fMRI studies exploring whether early-onset smokers present decreased activation in the anterior insular cortex during risk-processing tasks while responding to negative and positive interoceptive stimuli in comparison with late-onset smokers.Conducting fMRI studies exploring whether early-onset smokers present greater activation in the anterior insular cortex while exposed to smoking cues during tobacco withdrawal in comparison with late-onset smokers and correlate such activation with measures of tobacco craving .Findings from these studies may confirm whether early-onset smokers present a more compulsive smoking behavior in comparison with late-onset smokers during adulthood and may verify whether this compulsive smoking behavior is associated with structural and/or functional abnormalities in the anterior insular cortex. These results may inform the development of targeted brain stimulation treatments  aiming at preventing the transition from a reward-directed impulsive smoking behavior to compulsive tobacco-seeking and smoking in early-onset smokers.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the London Bromley Research Ethics Committee (REC) (REC Reference Number: 19/LO/1176) and by the University of St. Andrews Teaching and Research Ethics Committee (UTREC) . The patients/participants provided their written informed consent to participate in this study.AC was responsible for the conceptualization of the content and write-up of the current manuscript. AB provided supervision. Both authors critically reviewed the content and approved the final version for publication.


The 5-year survival rate for women with metastatic breast cancer is 29%. Potential biomarker identification is important for new treatment modalities in affected patients. Previous studies have shown that Parathyroid hormone-related peptide (PTHrP) plays a critical role in breast cancer growth and metastasis. Our study aimed to use a genetically modified breast cancer mouse model to precisely examine the role of PTHrP from early primary breast cancer initiation to late progression. We identified a novel long non-coding RNA (lncRNA), a new target for fatty acid metabolism that can be regulated via PTHrP in our unique mouse breast cancer model. We confirmed that a potential human lncRNA, OLMALINC, plays a similar role in fatty acid metabolism that can be regulated via PTHrP and validated our mouse findings in human breast cancer cell lines. Genetically engineered mouse models provide valuable tools to study the molecular metabolism for breast cancer progression.Parathyroid hormone-related peptide (PTHrP) is the primary cause of malignancy-associated hypercalcemia (MAH). We previously showed that PTHrP ablation, in the MMTV-PyMT murine model of breast cancer (BC) progression, can dramatically prolong tumor latency, slow tumor growth, and prevent metastatic spread. However, the signaling mechanisms using lineage tracing have not yet been carefully analyzed. Here, we generated Pthrpflox/flox; Cre+ mT/mG mice (KO) and Pthrpwt/wt; Cre+ mT/mG tumor mice (WT) to examine the signaling pathways under the control of PTHrP from the early to late stages of tumorigenesis. GFP+ mammary epithelial cells were further enriched for subsequent RNA sequencing (RNAseq) analyses. We observed significant upregulation of cell cycle signaling and fatty acid metabolism in PTHrP WT tumors, which are linked to tumor initiation and progression. Next, we observed that the expression levels of a novel lncRNA, GM50337, along with stearoyl-Coenzyme A desaturase 1 (Scd1) are significantly upregulated in PTHrP WT but not in KO tumors. We further validated a potential human orthologue lncRNA, OLMALINC, together with SCD1 that can be regulated via PTHrP in human BC cell lines. In conclusion, these novel findings could be used to develop targeted strategies for the treatment of BC and its metastatic complications. Nearly 30% of women initially diagnosed with early-stage breast cancer (BC) will present with metastases to distant organs, such as bones, lungs, liver, and brain, in addition to lymph nodes ,2. The 5PTHrP was initially identified as a humoral factor that underlies the development of hypercalcemia in malignancy, a severe complication in patients with advanced-stage cancers . It is eNext-generation sequencing (NGS) technology has revolutionized the field of cancer research over the last decade and a half. It unravels the fact that only about 2% of the human genome is annotated as protein-coding genes, whereas up to 98% of the transcriptional output is made of non-coding RNAs (ncRNAs) . Accordiflox/flox; Cre+ and Pthrpwt/wt; Cre+ mT/mG, PyMT BC mouse model, which allowed us to specifically trace and enrich the green fluorescent protein (GFP) mammary epithelial cells from early- to late-stage carcinoma. We applied comprehensive and unbiased transcriptome analyses to compare the gene expression profile between Pthrpwt/wt; Cre+ and Pthrpflox/flox; Cre+ tumors from three distinct stages of BC progression . We demonstrate that the disruption of PTHrP expression has a significant impact on tumor growth mainly via its regulation of gene expressions involved in several cancer hallmark signaling pathways, including cell cycle and metabolic adaptation. Moreover, we provide novel insights into the molecular mechanism of PTHrP as an oncogene that can activate a key enzyme, SCD1, for fatty acid metabolism via a cis-acting mechanism of a nearby lncRNA. Therefore, we propose that targeting these novel pathways driven by PTHrP can be beneficial in the treatment of BC.Our own work, using a mouse mammary tumor virus-polyoma middle tumor-antigen (MMTV-PyMT) oncoprotein mouse model of BC, has established that PTHrP is a key regulator of tumor initiation and progression . We alsoPthrp floxed mice (flox/floxPthrp) . Ta. TaGM503To assess the effects of the PTHrP on cellular lipid metabolism via OLMALINC gene expression, we treated two different human BC cell lines with a PTHrP monoclonal antibody (mAb) which was generated in our laboratory. In line with our mouse RNAseq results, a 70% to 80% decrease in OLMALINC expression following PTHrP mAb treatment for 72 h resulted in an approximately 70% decrease in SCD expression in both MDA-MB-231 and BT-549, respectively B. As a vhttp://gepia.cancer-pku.cn/ (accessed on 1 January 2022))  binding sites at its TSSs using ENCODE project data . SREBP1/y 2022))  45]. Us. Ushttp:y 2022)) C. We alsy 2022)) C. These We generated a mammary epithelial-specific Pthrp-knockout in PyMT mice in which mammary epithelial cells are specifically traced by membrane-targeted GFP. In line with our previous findings using the same BC mouse model, we observed genes that regulate cell cycle signaling were significantly upregulated by PTHrP as early as hyperplasia. We also demonstrated for the first time that PTHrP upregulates Scd1 encoding for the key fatty acid metabolism enzyme via the activation of a novel oncogenic lncRNA-GM50337. We further validated our findings in human BC cell lines demonstrating that PTHrP can regulate the transcription of human SCD via a candidate human lncRNA. Overall, we used an unbiased RNAseq approach to unravel all the transcriptomes altered via Pthrp ablation, including critical PCGs and novel lncRNAs regulating fatty acid metabolism. To investigate the involvement of PTHrP in primary BC initiation and progression, we generated a mammary epithelial-specific Pthrp-knockout  in a transgenic BC mouse model (PyMT) which Cre specifically expressed in mammary epithelium is monitored by membrane-targeted GFP (mT/mG reporter). During BC initiation, pre-neoplastic transformed epithelial cells only represent a small proportion of the total mammary epithelial population among all types of cells in the mammary gland. This advanced mouse model allowed us to specifically enrich total mammary epithelial populations, including both normal and pre-neoplastic cells for subsequent RNAseq analyses as early as hyperplasia. Therefore, we were able to specifically examine the transcriptomes of mammary epithelial lineage by comparing the tumor with tumor-free mice in order to identify the DEGs underlying epithelial phenotypes during tumor initiation using such a novel approach. Secondly, our previous studies have shown that lung metastasis from PTHrP KO mice was mainly derived from PTHrP-expressing tumor cells that escaped gene ablation . This isEven though this mouse model is an invaluable tool for our study, they have some limitations. Specifically, hierarchical clustering analyses indicated that the MMTV-PyMT mouse model is initially associated with the mature luminal signature characterized by the expression of luminal keratin 8/18, ER+, and PR+ at an early stage of tumor progression but progresses to luminal progenitor features characterized by basal-like tumors with low levels of ER in later stages, which is associated with metastasis . In contIn our study, we reported for the first time the transcriptional changes between PTHrP WT and KO tumors associated with three distinct stages in BC progression in mammary epithelial cells. Pthrp ablation was accompanied by a significant delay in tumor initiation characterized by the disruption of several hallmarks of cancer, including proliferation signaling and cellular metabolism. We observed that the ablation of Pthrp significantly downregulated genes involved in the cell cycle, including G2M checkpoint and E2F targets, supporting our previous finding using the same BC mouse model . E2F traImportantly, we observed, for the first time, using an unbiased genome-wide gene expression approach, that PTHrP can regulate fatty acid metabolism in BC early stage. Alterations of lipid metabolism have received increasing attention as a hallmark of cancer, and the dysregulation of its related enzymes has been linked to oncogenic activation and the maintenance of cancer cell survival and metastatic potential . SCD1 isOur work also provides additional support for future applications of anti-PTHrP therapy in clinical trials in patients with BC. Human BC cells treated with anti-PTHrP mAb in vitro had a remarkable decrease in growth . We alsoMoreover, our RNAseq data revealed that lncRNAs are abundantly differentially expressed in mammary tumors compared to normal mammary epithelial cells as early as the hyperplasia stage. Several lncRNAs identified in our study have been studied previously. One of these lncRNAs is Foxd2os, and the corresponding human counterpart is lncRNA FOXD2 adjacent to opposite strand RNA 1 (FOXD2-AS1). FOXD2-AS1 is aberrantly expressed in various human cancers, including BC, and is associated with cancer progression . MoreoveOur study emphasizes the importance of unbiased screening approaches and identifies novel signaling pathways that can be regulated via PTHrP. The majority of the differentially expressed lncRNAs in PTHrP WT tumors compared to the KO tumors have not been described previously. The ablation of Pthrp significantly downregulated lncRNA expression, which has the potential to cis-regulate nearby PCGs in both mouse primary BC cells (GM50337 and Scd1) and human BC cell lines (OLMALINC and SCD). Our results indicate that Pthrp-regulated lncRNAs are important mediators during BC progression. Our study provides novel evidence that PTHrP is an important regulator of lncRNA involving fatty acid metabolism and thereby plays a critical role in BC progression. RNAseq analysis often lacks systematic functional validation. Here, we demonstrated the identified PTHrP-associated GM50337 using both computational approaches and molecular assays. We performed an ASO-mediated knockdown of GM50337 and observed a significant reduction in the proliferation of primary mouse BC cells. This suggests that functional lncRNA transcripts contribute to the hallmarks of cancer and, therefore, are appealing potential therapeutic targets. However, the functions of lncRNAs in cancer biology are still elusive. Our identification of PTHrP-regulated mouse lncRNA and the study of its relevant functions also have some limitations. First, many lncRNAs in mice are not highly conserved in humans, which often hinders the identification of human orthologs via a mouse model. In our study, we only observed the conservation of mouse GM50337 at the level of genetic location in the human genome but not based on sequencing conservation. Thus, we looked for a potential human ortholog of mouse GM50337 based on its location rather than its sequence conservation. Synteny between lncRNAs and PCGs is frequently conserved across species . Future Our loss-of-function experiments in advanced genetically engineered mouse models of BC progression and in vitro human BC cells demonstrate that PTHrP deletion/inhibition results in a significant BC initiation delay via the interruption of cell growth and fatty acid metabolism remodeling. We propose that targeting these novel pathways, including these lncRNAs controlled by PTHrP, could be used to develop targeted therapeutic strategies in BC and its metastatic complications.