Specific inhibition of an anticancer target, polo-like kinase 1, by allosterically dismantling its mechanism of substrate recognition.

Polo-like kinase 1 (Plk1) is considered an attractive target for anticancer therapy. Over the years, studies on the noncatalytic polo-box domain (PBD) of Plk1 have raised the expectation of generating highly specific protein-protein interaction inhibitors. However, the molecular nature of the canonical PBD-dependent interaction, which requires extensive water network-mediated interactions with its phospholigands, has hampered efforts to identify small molecules suitable for Plk1 PBD drug discovery. Here, we report the identification of the first allosteric inhibitor of Plk1 PBD, called Allopole, a prodrug that can disrupt intracellular interactions between PBD and its cognate phospholigands, delocalize Plk1 from centrosomes and kinetochores, and induce mitotic block and cancer cell killing. At the structural level, its unmasked active form, Allopole-A, bound to a deep Trp-Phe-lined pocket occluded by a latch-like loop, whose adjoining region was required for securely retaining a ligand anchored to the phospho-binding cleft. Allopole-A binding completely dislodged the L2 loop, an event that appeared sufficient to trigger the dissociation of a phospholigand and inhibit PBD-dependent Plk1 function during mitosis. Given Allopole's high specificity and antiproliferative potency, this study is expected to open an unexplored avenue for developing Plk1 PBD-specific anticancer therapeutic agents.

A multiplex real-time RT-qPCR assay for simultaneous detection of porcine epidemic diarrhea virus, porcine deltacoronavirus, and swine acute diarrhea syndrome coronavirus.

Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV) cause intestinal diseases with similar manifestations in suckling piglets. In this study, we developed a multiplex real-time PCR for differential diagnosis of PEDV, PDCoV, and SADS-CoV. The assay demonstrated high specificity with a detection limit of 5 copies/µl for each virus. The assay specifically detected PEDV, PDCoV, and SADS-CoV and excluded all other swine pathogens circulating in pigs. Furthermore, the assay exhibited satisfactory performance in analyzing clinical samples. The data indicate that the newly developed multiplex real-time PCR method can be applied for differential diagnosis of porcine enteric coronaviruses.

Relation Between Masticatory Performance and Skeletal Properties in Patients With Skeletal Class III Malocclusion.

This study aimed to measure masticatory performance (MP) using ß-carotene gummy jelly to investigate its relationship with skeletal properties in decompensated patients diagnosed with skeletal class III malocclusion. The study included 78 patients (38 men and 40 women) diagnosed with skeletal class III malocclusion without temporomandibular joint disorder and periodontal disease. MP was measured using a new masticatory measuring device and ß-carotene in the gummy jelly. Lateral and posteroanterior cephalograms were obtained, and skeletal properties (Me deviation, ANB, SNB, APDI, Wits, ODI, facial axis, body length, ramus length, SN-GoGn, anterior facial height, posterior facial height, saddle angle, articular angle, and gonial angle) were evaluated. MP differences according to age and sex and the effect of skeletal properties on MP were analyzed using multiple linear regression analysis. The MP of all patients was 3690.55±1428.77 mm², MP of the male group was 4043.05±1498.09 mm², and MP of the female group was 3355.68±1272.19 mm². Among the items investigated, the variable that affected MP was posterior facial height. Posterior facial height showed a positive correlation (P=0.022). There was no significant difference between MP and other skeletal properties (P>0.05). The severity of the hypodivergency in skeletal class III could affect MP. The relationship between facial asymmetry or skeletal relation and MP could not be explained in this study.

Early molecular events of autosomal-dominant Alzheimer's disease in marmosets with PSEN1 mutations.

INTRODUCTION: Fundamental questions remain about the key mechanisms that initiate Alzheimer's disease (AD) and the factors that promote its progression. Here we report the successful generation of the first genetically engineered marmosets that carry knock-in (KI) point mutations in the presenilin 1 (PSEN1) gene that can be studied from birth throughout lifespan. METHODS: CRISPR/Cas9 was used to generate marmosets with C410Y or A426P point mutations in PSEN1. Founders and their germline offspring are comprehensively studied longitudinally using non-invasive measures including behavior, biomarkers, neuroimaging, and multiomics signatures. RESULTS: Prior to adulthood, increases in plasma amyloid beta were observed in PSEN1 mutation carriers relative to non-carriers. Analysis of brain revealed alterations in several enzyme-substrate interactions within the gamma secretase complex prior to adulthood. DISCUSSION: Marmosets carrying KI point mutations in PSEN1 provide the opportunity to study the earliest primate-specific mechanisms that contribute to the molecular and cellular root causes of AD onset and progression. HIGHLIGHTS: We report the successful generation of genetically engineered marmosets harboring knock-in point mutations in the PSEN1 gene. PSEN1 marmosets and their germline offspring recapitulate the early emergence of AD-related biomarkers. Studies as early in life as possible in PSEN1 marmosets will enable the identification of primate-specific mechanisms that drive disease progression.

Deletion of hepatic small heterodimer partner ameliorates development of nonalcoholic steatohepatitis in mice.

Small heterodimer partner (SHP, Nr0b2) is an orphan nuclear receptor that regulates bile acid, lipid, and glucose metabolism. Shp-/- mice are resistant to diet-induced obesity and hepatic steatosis. In this study, we explored the potential role of SHP in the development of nonalcoholic steatohepatitis (NASH). A 6-month Western diet (WD) regimen was used to induce NASH. Shp deletion protected mice from NASH progression by inhibiting inflammatory and fibrotic genes, oxidative stress, and macrophage infiltration. WD feeding disrupted the ultrastructure of hepatic mitochondria in WT mice but not in Shp-/- mice. In ApoE-/- mice, Shp deletion also effectively ameliorated hepatic inflammation after a 1 week WD regimen without an apparent antisteatotic effect. Moreover, Shp-/- mice resisted fibrogenesis induced by a methionine- and choline-deficient diet. Notably, the observed protection against NASH was recapitulated in liver-specific Shp-/- mice fed either the WD or methionine- and choline-deficient diet. Hepatic cholesterol was consistently reduced in the studied mouse models with Shp deletion. Our data suggest that Shp deficiency ameliorates NASH development likely by modulating hepatic cholesterol metabolism and inflammation.

Microbial products linked to steatohepatitis are reduced by deletion of nuclear hormone receptor SHP in mice.

Deletion of the nuclear hormone receptor small heterodimer partner (Shp) ameliorates the development of obesity and nonalcoholic steatohepatitis (NASH) in mice. Liver-specific SHP plays a significant role in this amelioration. The gut microbiota has been associated with these metabolic disorders, and the interplay between bile acids (BAs) and gut microbiota contributes to various metabolic disorders. Since hepatic SHP is recognized as a critical regulator in BA synthesis, we assessed the involvement of gut microbiota in the antiobesity and anti-NASH phenotype of Shp-/- mice. Shp deletion significantly altered the levels of a few conjugated BAs. Sequencing the 16S rRNA gene in fecal samples collected from separately housed mice revealed apparent dysbiosis in Shp-/- mice. Cohousing Shp-/- mice with WT mice during a Western diet regimen impaired their metabolic improvement and effectively disrupted their distinctive microbiome structure, which became indistinguishable from that of WT mice. While the Western diet challenge significantly increased lipopolysaccharide and phenylacetic acid (PAA) levels in the blood of WT mice, their levels were not increased in Shp-/- mice. PAA was strongly associated with hepatic peroxisome proliferator-activated receptor gamma isoform 2 (Pparg2) activation in mice, which may represent the basis of the molecular mechanism underlying the association of gut bacteria and hepatic steatosis. Shp deletion reshapes the gut microbiota possibly by altering BAs. While lipopolysaccharide and PAA are the major driving forces derived from gut microbiota for NASH development, Shp deletion decreases these signaling molecules via dysbiosis, thereby partially protecting mice from diet-induced metabolic disorders.

Adsorption Capacity and Desorption Efficiency of Activated Carbon for Odors from Medical Waste.

Five types of odor-emitting exhaust gases from medical waste were selected, and their adsorption capacity and desorption efficiency were investigated using activated carbon. The selected gases included polar gases (hydrogen sulfide (H2S) and ammonia (NH3)) and non-polar gases (acetaldehyde (AA), methyl mercaptan (MM), and trimethylamine (TMA))). Commercial activated carbon with a specific surface area of 2276 m2/g was used as the adsorbent. For the removal of odor from medical waste, we investigated: (1) the effective adsorption capacity of a single gas (<1 ppm), (2) the effect of the adsorbed NH3 gas concentration and flow rate, and (3) the desorption rate using NH3 gas. The values of the effective adsorption capacity of the single gas were in the following order: H2S < NH3 < AA < MM < TMA, at 0.2, 4.2, 6.3, 6.6, and 35.7 mg/g, respectively. The results indicate that polar gases have a lower effective adsorption capacity than that of non-polar gases, and that the size of the gas molecules and effective adsorption capacity exhibit a proportional relationship. The effective adsorption performance of NH3 gas showed an increasing trend with NH3 concentration. Therefore, securing optimal conditions for adsorption/desorption is imperative for the highly efficient removal of odor from medical waste.

A chimeric MERS-CoV virus-like particle vaccine protects mice against MERS-CoV challenge.

BACKGROUND: Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory disease in humans, with a case fatality rate of approximately 35%, thus posing a considerable threat to public health. The lack of approved vaccines or antivirals currently constitutes a barrier in controlling disease outbreaks and spread. METHODS: In this study, using a mammalian expression system, which is advantageous for maintaining correct protein glycosylation patterns, we constructed chimeric MERS-CoV virus-like particles (VLPs) and determined their immunogenicity and protective efficacy in mice. RESULTS: Western blot and cryo-electron microscopy analyses demonstrated that MERS-CoV VLPs were efficiently produced in cells co-transfected with MERS-CoV spike (S), envelope, membrane and murine hepatitis virus nucleocapsid genes. We examined their ability as a vaccine in a human dipeptidyl peptidase 4 knock-in C57BL/6 congenic mouse model. Mice immunized with MERS VLPs produced S-specific antibodies with virus neutralization activity. Furthermore, MERS-CoV VLP immunization provided complete protection against a lethal challenge with mouse-adapted MERS-CoV and improved virus clearance in the lung. CONCLUSIONS: Overall, these data demonstrate that MERS-CoV VLPs have excellent immunogenicity and represent a promising vaccine candidate.

Anti-Inflammatory and Neuroprotective Mechanisms of GTS-21, an α7 Nicotinic Acetylcholine Receptor Agonist, in Neuroinflammation and Parkinson's Disease Mouse Models.

Neuroinflammation is crucial in the progression of neurodegenerative diseases. Thus, controlling neuroinflammation has been proposed as an important therapeutic strategy for neurodegenerative disease. In the present study, we examined the anti-inflammatory and neuroprotective effects of GTS-21, a selective α7 nicotinic acetylcholine receptor (α7 nAChR) agonist, in neuroinflammation and Parkinson's disease (PD) mouse models. GTS-21 inhibited the expression of inducible nitric oxide synthase (iNOS) and proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated BV2 microglial cells and primary microglia. Further research revealed that GTS-21 has anti-inflammatory properties by inhibiting PI3K/Akt, NF-κB, and upregulating AMPK, Nrf2, CREB, and PPARγ signals. The effects of GTS-21 on these pro-/anti-inflammatory signaling molecules were reversed by treatment with an α7 nAChR antagonist, suggesting that the anti-inflammatory effects of GTS-21 are mediated through α7 nAChR activation. The anti-inflammatory and neuroprotective properties of GTS-21 were then confirmed in LPS-induced systemic inflammation and MPTP-induced PD model mice. In LPS-injected mouse brains, GTS-21 reduced microglial activation and production of proinflammatory markers. Furthermore, in the brains of MPTP-injected mice, GTS-21 restored locomotor activity and dopaminergic neuronal cell death while inhibiting microglial activation and pro-inflammatory gene expression. These findings suggest that GTS-21 has therapeutic potential in neuroinflammatory and neurodegenerative diseases such as PD.

Severe Fever with Thrombocytopenia Syndrome Virus in Ticks in the Republic of Korea.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a zoonotic, tick-borne RNA virus of the genus Bandavirus (Family Phenuiviridae), mainly reported in China, Japan, and the Republic of Korea (Korea). For the purpose of this study, a total of 3,898 adult and nymphal ticks of species Haemaphysalis longicornis (94.2%), Haemaphysalis flava (5.0%), Ixodes nipponensis (0.8%), and 1 specimen of Ixodes ovatus, were collected from the Deogyusan National Park, Korea, between April 2016 and June 2018. A single-step reverse transcriptase-nested PCR was performed, targeting the S segment of the SFTSV RNA. Total infection rate (IR) of SFTSV in individual ticks was found to be 6.0%. Based on developmental stages, IR was 5.3% in adults and 6.0% in nymphs. The S segment sequences obtained from PCR were divided into 17 haplotypes. All haplotypes were phylogenetically clustered into clades B-2 and B-3, with 92.7% sequences in B-2 and 7.3% in B-3. These observations indicate that the Korean SFTSV strains were closer to the Japanese than the Chinese strains. Further epidemiological studies are necessary to better understand the characteristics of the Korean SFTSV and its transmission cycle in the ecosystem.

Dynamic expression of tRNA-derived small RNAs define cellular states.

Transfer RNA (tRNA)-derived small RNAs (tsRNAs) have recently emerged as important regulators of protein translation and shown to have diverse biological functions. However, the underlying cellular and molecular mechanisms of tsRNA function in the context of dynamic cell-state transitions remain unclear. Expression analysis of tsRNAs in distinct heterologous cell and tissue models of stem vs. differentiated states revealed a differentiation-dependent enrichment of 5'-tsRNAs. We report the identification of a set of 5'-tsRNAs that is upregulated in differentiating mouse embryonic stem cells (mESCs). Notably, interactome studies with differentially enriched 5'-tsRNAs revealed a switch in their association with "effector" RNPs and "target" mRNAs in different cell states. We demonstrate that specific 5'-tsRNAs can preferentially interact with the RNA-binding protein, Igf2bp1, in the RA-induced differentiated state. This association influences the transcript stability and thereby translation of the pluripotency-promoting factor, c-Myc, thus providing a mechanistic basis for how 5'-tsRNAs can modulate stem cell states in mESCs. Together our study highlights the role of 5'-tsRNAs in defining distinct cell states.

Heat shock protein 70 could enhance porcine epidemic diarrhoea virus replication by interacting with membrane proteins.

In this study, we investigated the role of heat shock protein 70 (HSP70) in porcine epidemic diarrhoea virus (PEDV) replication. We found that PEDV infection induced strong HSP70 overexpression in the very early stage of infection. We also confirmed that HSP70 overexpression increased the speed of PEDV replication, resulting in the generation of more virions. In contrast, knockout of HSP70 in cells significantly downregulated PEDV protein expression, resulting in a significant reduction in PEDV replication. Most importantly, we confirmed that among the structural proteins of PEDV, membrane (M) proteins have this important role. We found that membrane proteins control cellular HSP70 expression in PEDV-infected cells. We confirmed HSP70/M complex formation by both immunoprecipitation and immunofluorescence assays. Additionally, PEDV M overexpression induced strong HSP70 expression. All our results clearly confirmed that in PEDV-infected cells, the M protein plays a very important role in PEDV replication in collaboration with HSP70.

CGFS-type glutaredoxin mutations reduce tolerance to multiple abiotic stresses in tomato.

Sessile organisms such as plants have adopted diverse reactive oxygen species (ROS) scavenging mechanisms to mitigate damage under abiotic stress conditions. Though CGFS-type glutaredoxin (GRX) genes are important regulators of ROS homeostasis, each of their functions in crop plants have not yet been well understood. We performed a targeted mutagenesis analysis of four CGFS-type GRXs (SlGRXS14, SlGRXS15, SlGRXS16, and SlGRXS17) in tomato plants (Solanum lycopersicum) using a multiplex clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system and found that Slgrxs mutants were more sensitive to various abiotic stresses compared with the wild-type tomatoes. Slgrxs15 mutants were embryonic lethal. Single, double, and triple combinations of Slgrxs14, 16, and 17 mutants were examined under heat, chilling, drought, heavy metal toxicity, nutrient deficiency, and short photoperiod stresses. Slgrxs14 and 17 mutants showed hypersensitivity to almost all stresses while Slgrxs16 mutants were affected by chilling stress and showed milder sensitivity to other stresses. Additionally, Slgrxs14 and 17 mutants showed delayed flowering time. Our results indicate that the CGFS-type SlGRXs have specific roles against abiotic stresses, providing valuable resources to develop tomato and, possibly, other crop species that are tolerant to multiple abiotic stresses by genetic engineering.

Inhibitory Neural Network's Impairments at Hippocampal CA1 LTP in an Aged Transgenic Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a rapid accumulation of amyloid ß (Aß) protein in the hippocampus, which impairs synaptic structures and neuronal signal transmission, induces neuronal loss, and diminishes memory and cognitive functions. The present study investigated the impact of neuregulin 1 (NRG1)-ErbB4 signaling on the impairment of neural networks underlying hippocampal long-term potentiation (LTP) in 5xFAD mice, a model of AD with greater symptom severity than that of TG2576 mice. Specifically, we observed parvalbumin (PV)-containing hippocampal interneurons, the effect of NRG1 on hippocampal LTP, and the functioning of learning and memory. We found a significant decrease in the number of PV interneurons in 11-month-old 5xFAD mice. Moreover, synaptic transmission in the 5xFAD mice decreased at 6 months of age. The 11-month-old transgenic AD mice showed fewer inhibitory PV neurons and impaired NRG1-ErbB4 signaling than did wild-type mice, indicating that the former exhibit the impairment of neuronal networks underlying LTP in the hippocampal Schaffer-collateral pathway. In conclusion, this study confirmed the impaired LTP in 5xFAD mice and its association with aberrant NRG1-ErbB signaling in the neuronal network.

Oxidative Damage to the TCA Cycle Enzyme MDH1 Dysregulates Bioenergetic Enzymatic Activity in the Aged Murine Brain.

Aging can have profound effects on the mammalian brain leading to neurodegeneration and cognitive impairment. The brain has exceptionally high-energy requirements and is particularly susceptible to damage within its bioenergetic pathways. Here, we asked how the bioenergetic proteome of the murine brain changed with age and how this might affect brain function. Using label-free LC-MS/MS proteomics for the discovery phase and quantitative multiple reaction monitoring LC-MRM-MS/MS for the validation phase, we found dysregulated expression of multiple components of the tricarboxylic acid cycle, which is key for mitochondrial energy production, including SULA2, IDH1, IDH2, SDHB, PDHB, MDH1, FH1, and NDUFS3, in old murine brains. We also saw that the oxidoreductases, thioredoxin and glutaredoxin, were significantly down-regulated in the old mouse brain and showed through MS that this correlated with the accumulation of trioxidation in the key metabolic enzyme MDH1 at Cys137. 3D modeling of MDH1 predicted that the damaged sites were located at the protein active zone, and enzymatic kinetic analysis confirmed that MDH1 function was significantly reduced in the old mouse brain. These findings identify the tricarboxylic acid cycle as a key target of degenerative protein modifications with deleterious effects on the aging brain's bioenergetic function.

Tissue-specific roles of GCN2 in aging and autosomal dominant retinitis pigmentosa.

The organisms have the capacity to sense and adapt to their surroundings for their life in a dynamic environment. In response to amino acid starvation, cells activate a rectifying physiological program, termed the integrated stress response (ISR), to restore cellular homeostasis. General controlled non-repressed (GCN2) kinase is a master regulator of the ISR and modulates protein synthesis in response to amino acid starvation. We previously established the GCN2/ATF4/4E-BP pathway in development and aging. Here, we investigated the tissue-specific roles of GCN2 upon dietary restriction of amino acid in a Drosophila model. The knockdown of GCN2 in the gut and fat body, an energy sensing organ in Drosophila, abolished the beneficial effect of GCN2 in lifespan extension upon dietary restriction of amino acids. Proteome analysis in an autosomal dominant retinitis pigmentosa (ADRP) model showed that dietary restriction of amino acids regulates the synthesis of proteins in several pathways, including mitochondrial translation, mitochondrial gene expression, and regulation of biological quality, and that gcn2-mutant flies have reduced levels of these mitochondria-associated proteins, which may contribute to retinal degeneration in ADRP. These results indicate that the tissue-specific regulation of GCN2 contributes to normal physiology and ADRP progression.

Development of a Squaraine-Based Molecular Probe for Dual-Modal in Vivo Fluorescence and Photoacoustic Imaging.

Dual-modular imaging approaches combining near-infrared (NIR) fluorescence (FLI) and photoacoustic imaging (PAI) require suitable contrast agents to produce dual-modular signals. Although nanoparticles have been used to develop PAI agents, small molecule-based imaging agents have not been extensively studied, highlighting the need to design new fluorophores with an enhanced multifunctional ability. Thus, in this study, we designed a novel squaraine (SQ)-based dye and reported its rational preparation and conjugation with a cancer targeting peptide. Specifically, benzoindole-derived SQ (BSQ) showed strong absorption and fluorescence properties at above 650 nm under aqueous conditions, with a maximum absorption and emission at 665 and 680 nm, respectively. Moreover, PA signal scanning experiments revealed a maximum signal intensity in the range 680-700 nm. BSQ was also conjugated with cyclic arginine-glycine-aspartic acid (cRGD) to improve its active targeting ability for the αvß3 integrin, which is overexpressed in various cancer and angiogenic cells. A series of in vitro, in vivo, and ex vivo FLI studies showed that the cRGD conjugated BSQ (BSQ-RGD2) successfully stained and targeted αvß3 integrin-overexpressing tumor cells and xenografts, which were clearly visualized by FLI and PAI. Therefore, BSQ-RGD2 can successfully be applied to dual-modular imaging of the specific biomarker in living animals.

Alpha-neoendorphin can reduce UVB-induced skin photoaging by activating cellular autophagy.

Skin aging is influenced by several genetic, physiological, and environmental factors. In particular, ultraviolet (UV) exposure is an important factor involved in inducing skin photoaging. Autophagy controlling homeostatic balance between the synthesis, degradation, and recycling of cellular organelles and proteins plays important regulatory roles in several biological processes, including aging. The opioid neuropeptide α-neoendorphin (named NEP) is an endogenous decapeptide (N-YGGFLRKYPK-C) that activates the kappa opioid receptor and exhibits certain anti-aging and anti-wrinkling effects on skin cells; however, its action mechanism has not yet been elucidated. Therefore, the aim of this study was to determine the effects of NEP on anti-skin aging and autophagy activation in human dermal fibroblast cells. Western blot results showed that NEP down-regulates the production of phospho-mammalian target of rapamycin (p-mTOR), whereas increases the expression of key autophagy-related molecules such as Beclin-1, Atg5-Atg12, and LC3-II. The immunocytochemical analysis performed with anti-LC3-II antibody also showed that the autophagic indicators, autophagosomes are formed by NEP. These results suggest that NEP can activate cellular autophagy through mTOR-Beclin-1-mediated signaling pathway. It was also revealed by CM-H2DCF-DA assay and Western blottings that NEP can reduce the production of ultraviolet B (UVB)-induced reactive oxygen species (ROS) like with N-acetylcysteine (NAC), resulting in decreasing the expression levels of skin aging-related proteins, such as phospho-ERK (p-ERK), phospho-p38 (p-p38), and phospho-JNK (p-JNK). Furthermore, NEP could increase the type I procollagen production, while decreasing MMP-1, MMP-2, and MMP-9 activities. Taken together, the results demonstrate that NEP can reduce UVB-induced photoaging by activating autophagy.

Application of Ultrasound Thermal Imaging for Monitoring Laser Ablation in Ex Vivo Cardiac Tissue.

BACKGROUND AND OBJECTIVE: Laser ablation can be used to treat atrial fibrillation by thermally isolating pulmonary veins. In this study, we evaluated the feasibility of high-resolution (<1 mm) ultrasound thermal imaging to monitor spatial temperature distribution during laser ablation on ex vivo cardiac tissue. STUDY DESIGN/MATERIALS AND METHODS: Laser ablation (808 nm) was performed on five porcine cardiac tissue samples. A thermocouple was used to measure the interstitial tissue temperature during the laser ablation process. Tissue-strain-based ultrasound thermal imaging was conducted to monitor the spatial distribution of the temperature in the cardiac tissue. The tissue temperature was estimated from the time shifts of ultrasound signals owing to the changes in the speed of sound and was compared with the measured temperature. The temperature estimation coefficient k of porcine cardiac tissue was calculated from the estimated thermal strain and the measured temperature. The degree of tissue coagulation (temperatures > 50°C) was derived from the estimated temperature and was compared with that of the tested cardiac tissue. RESULTS: The estimated tissue temperature using strain-based ultrasound thermal imaging at a depth of 1 mm agreed with thermocouple measurements. During the 30-second period of the laser ablation process, the estimated tissue temperature increased from 25 to 70°C at a depth of 0.1 mm, while the estimated temperature at a depth of 1 mm increased up to 46°C. Owing to the uncertainty of the coefficient k, the k value of the porcine cardiac tissue varied from 160 to 220°C with temperature changes of up to 20°C. The estimated coagulation region in the ultrasound thermal imaging was 20% wider (+0.6 mm) but 9% shallower (-0.1 mm) than the measured region of the ablated porcine cardiac tissue. CONCLUSIONS: The current study demonstrated the feasibility of temperature monitoring with the use of ultrasound thermal imaging during the laser ablation on ex vivo porcine cardiac tissue. The high-resolution ultrasound thermal imaging could map the spatial distribution of the tissue temperature. The proposed method can be used to monitor the temperature and thermal coagulation to achieve effective laser ablation for atrial fibrillation. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Mouse-adapted MERS coronavirus causes lethal lung disease in human DPP4 knockin mice.

The Middle East respiratory syndrome (MERS) emerged in Saudi Arabia in 2012, caused by a zoonotically transmitted coronavirus (CoV). Over 1,900 cases have been reported to date, with ∼36% fatality rate. Lack of autopsies from MERS cases has hindered understanding of MERS-CoV pathogenesis. A small animal model that develops progressive pulmonary manifestations when infected with MERS-CoV would advance the field. As mice are restricted to infection at the level of DPP4, the MERS-CoV receptor, we generated mice with humanized exons 10-12 of the mouse Dpp4 locus. Upon inoculation with MERS-CoV, human DPP4 knockin (KI) mice supported virus replication in the lungs, but developed no illness. After 30 serial passages through the lungs of KI mice, a mouse-adapted virus emerged (MERSMA) that grew in lungs to over 100 times higher titers than the starting virus. A plaque-purified MERSMA clone caused weight loss and fatal infection. Virus antigen was observed in airway epithelia, pneumocytes, and macrophages. Pathologic findings included diffuse alveolar damage with pulmonary edema and hyaline membrane formation associated with accumulation of activated inflammatory monocyte-macrophages and neutrophils in the lungs. Relative to the parental MERS-CoV, MERSMA viruses contained 13-22 mutations, including several within the spike (S) glycoprotein gene. S-protein mutations sensitized viruses to entry-activating serine proteases and conferred more rapid entry kinetics. Recombinant MERSMA bearing mutant S proteins were more virulent than the parental virus in hDPP4 KI mice. The hDPP4 KI mouse and the MERSMA provide tools to investigate disease causes and develop new therapies.