Mechanism of acoustic pressure spectrum shifting toward lower frequencies in applied current thermoacoustic imaging.

Objective. Thermoacoustic tomography (TAT) is a promising imaging technique used for early cancer diagnosis, tumor therapy, animal study and brain imaging. Although it is widely known that the TAT frequency response depends on the pulse width of the source and the size of the object, a thorough comprehension of the quantitative frequency modulation in TAT and the mechanism governing the shift in the thermoacoustic pressure spectrum towards lower frequencies with respect to the excitation source is still lacking. This study aims to understand why the acoustic pressure spectrum and the final voltage signals shift towards lower frequencies in TAT.Approach. We employed a linear time-invariant model. In the proposed model, the applied current thermoacoustic imaging (ACTAI) process is divided into the thermoacoustic stage and the acoustoelectric stage. These two stages are characterized by the thermoacoustic transfer function(TATF) and the transducer transfer function (TDTF), respectively. We confirmed the effectiveness of our model through a rigorous examination involving both simulations and experiments.Main results. Simulation results indicate that the TATF behaves as a low-pass filter. The inherent low-pass nature induces a shift towards low frequencies in the acoustic pressure spectrum. Experiments further confirm this behavior, demonstrating that the final electrical voltage also shifts towards low frequencies. Notably, employing the proposed model, there is a remarkable consistency between the main frequency bands of the synthesized and measured final voltage spectrum.Significance. The proposed model thoroughly explains how the TATF causes shifts to low frequencies in both the acoustic pressure spectrum and the final voltage spectrum in TAT. These insights deepen our understanding of optimizing TAT systems in the frequency domain, including aspects like filter design and transducer selection. Furthermore, we underscore the potential significance of this discovery for medical applications, particularly in the context of cancer diagnosis.

Homologous proteins SAPCD2X1 and SAPCD2 have significantly different carcinogenic capacities in human colorectal cancer cells based on structural prediction and functional verification.

We discussed the expression and biological functions of the SAPCD2X1 protein in the HCT116 CRC cell line by bioinformatics analysis and prediction, and biological function verification. Spatial conformation models of SAPCD2X1 and SAPCD2 were predicted using the threading method, ensemble method, and several other protein structure prediction approaches. The conformational similarity between SAPCD2X1 and SAPCD2 was studied, and their functions were predicted. The biological experiments showed that SAPCD2X1 and SAPCD2 were overexpressed in CRC cells. SAPCD2X1-specific antibodies were prepared. The expressions of SAPCD2X1 and SAPCD2 were localized in cells using the immunofluorescence assay. The SAPCD2 and SAPCD2X1 overexpression models were validated using Western Blot and RT-qPCR. We successfully predicted the structures of the SAPCD2X1 and SAPCD2 proteins, and visualized them using the VDM software. It was predicted that the tertiary structure of SAPCD2X1 changed significantly compared with SAPCD2. Alteration of the biological functions of SAPCD2X1 was also predicted due to the changes in the spatial conformation of the protein. Anti-SAPCD2X1 antibody and SAPCD2X1-EGFP and SAPCD2-EGFP recombinant plasmids were established. The overexpression of the two proteins was induced in HCT116 cells using the recombinant plasmids, and verified by RT-qPCR and Western Blot. Meanwhile, the anti-SAPCD2X1 antibody was proved to have a high specificity. The immunofluorescence assay showed that SAPCD2X1 and SAPCD2 are mainly expressed in the cytoplasm. SAPCD2X1 and SAPCD2 exhibited significantly different biological functions in HCT116 cells. SAPCD2 is a carcinogenic protein, while SAPCD2X1 does not affect the proliferation, invasion, and migration of human CRC HCT116 cells.

Periampullary tumors in a patient with pancreatic divisum and neurofibromatosis type 1: a case report.

INTRODUCTION: We present a case of a male patient with neurofibromatosis type 1 diagnosed with pancreatic divisum and several gastrointestinal tumors. A 55-year-old man was admitted to the hospital with recurrent chronic pancreatitis, indicating a large mass in the ampulla. In addition, genetic testing revealed two unique germline mutations in the neurofibromin (NF1) gene, and their potential interaction in promoting cancer was further investigated. CONCLUSION: The first similar case was reported in 2020. The current case was distinct from other cases since an additional two NF1 mutations were found in the patient. In conjunction with prior case reports, our findings imply that genetic testing in patients diagnosed with neurofibromatosis type 1 could be helpful in the development of effective treatments.

The Generation and Application of Monoclonal Antibodies to Detect SAPCD2 Expression in Precancerous and Malignant Gastric Lesions.

Background: Suppressor APC domain containing 2 (SAPCD2) is involved in cell cycle regulation and its mRNA levels are higher in cancer tissues. But, the function of SAPCD2 in cancer development remains unclear. Objective: To generate mouse monoclonal antibodies (mAbs) specific to SAPCD2 and thus clarify the function of SAPCD2 in the development of gastric carcinoma (GC). Methods: Purified SAPCD2-GST immunized BALB/c mouse spleen cells were collected and fused with myeloma cells to obtain monoclonal antibody hybridoma. A group of monoclonal antibodies exhibiting high specificity and sensitivity against SAPCD2 has been generated and characterized by IHC, WB, IP, IF, and ELISA. By immunohistochemical analysis, the SAPCD2 expression was evaluated in 228 clinical samples of gastric mucosal lesions, including precancerous lesions and GC samples. Results: We identified a highly specific and sensitive clone of s12 in eukaryotic cells and performed an IHC analysis. We found that 81.3% of 107 GC tissues were SAPCD2-positive, compared with the 26.2% in the matched adjacent normal-appearing tissues (P<0.001). Furthermore, among the 121 gastritis tissues, SAPCD2 was overexpressed in precancerous gastric lesions such as dysplasia (Dys, 78.9%), intestinal metaplasia (IM, 44.7%), and chronic atrophic gastritis (CAG, 6.1%) compared with that in chronic non-atrophic gastritis (CNAG, 3.2%) (P<0.001). The SAPCD2-positivity rate was 81.3% in GC, suggesting that the expression of SAPCD2 increased with the severity of the lesion (P<0.001). Conclusion: In summary, we have described novel monoclonal antibodies against SAPCD2, which are highly expressed in GC tissues and may serve as the basis for an early clinical marker for GC development.

Liquid metal injected from interstitial channels for inhibiting subcutaneous hepatoma growth and improving MRI/MAT image contrast.

Objective: Liquid metal (LM) nowadays is considered a new biomedical material for medical treatment. The most common application of LM in medical therapy is taking LM as a carrier for oncology therapeutics. However, the feasibility and direct effect of LM in tumor treatment are still unknown, and how to delineate the negative resection margin (NRM) of the tumor is also a crucial problem in surgery. We aimed to inject LM into interstitial channels of extremities of mice to overlay the surface of the primary tumor to investigate the effect of LM on inhibiting tumor growth and highlight the NRM of the tumor. Methods: In this study, all 50 BALB/c-nude female mice were used to construct the transplanted HepG2-type hepatocellular carcinoma model. One week after the establishment of the model, the mice were divided into three groups, named LM group, PBS group and Control group by injecting different liquid materials into the forelimb interstitial channel of the mice. T2WI image on MRI and Magneto-acoustic tomography (MAT) were used to show the distribution of LM and PBS in vivo. The group comparisons of tumor growth and blood tests were evaluated by one-way ANOVA and post-hoc analysis. And the biocompatibility of LM to BALB/c nude mice was evaluated by histopathological analysis of LM group and control group. Results: The volume change ratio of tumor was significantly lower in LM group than in PBS and Control group after 10 days of grouping. Compared with PBS and Control group, the main indexes of blood tests in LM group were significantly lower and close to normal level. In addition, the distribution of LM in vivo could be clearly observed under T2WI anatomic images and the crossprofile of the tumor in MAT. LM also has a obvious contrast in MRI T2WI and enhanced the amplitude of imaging signal in MAT. Conclusion: LM may inhibit the growth of transplanted hepatoma tumor through tumor encapsulation. In vivo, tumor imaging and LM distribution imaging were achieved by MRI T2WI, which verified that LM injected with interstitial injection made the NRM of tumor more prominent and had the potential of being MRI contrast agent. At the same time, LM could also be a new conductive medium to improve the imaging quality of MAT. Moreover, LM performed mild biocompatibility.

Associations of cortical iron accumulation with cognition and cerebral atrophy in Alzheimer's disease.

Background: In Alzheimer's disease (AD), cerebral iron accumulation colocalizes with the pathological proteins amyloid-ß (Aß) and tau. Furthermore, tau-induced cortical thinning is associated with cognitive decline. In this study, quantitative susceptibility mapping (QSM) was used to investigate the whole-brain distribution pattern of cortical iron deposition and its relationships with cognition and cortical thickness in AD. Methods: This cross-sectional study prospectively recruited 30 participants with AD and 26 age- and sex-matched healthy controls (HCs). All participants underwent QSM and T1-weighted examinations on a 3.0T MRI scanner. Global cognition was assessed using the Mini-Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA). Whole-brain cross-sectional QSM analysis and whole-brain QSM regression analyses against the MMSE and MoCA scores were performed. Surface-based morphometry analysis was also performed. Subsequently, in regions with significant atrophy, magnetic susceptibility was compared between the AD and HC groups, and the association between magnetic susceptibility and cortical thickness was assessed. Results: Whole-brain QSM cross-sectional analysis in the AD group demonstrated widespread increased susceptibility across the cortical ribbon, asymmetrically covering the left hemisphere cerebral cortex, caudate nucleus, putamen, and partial cerebellar cortex. Whole-brain QSM regression analyses in the AD group showed that increased susceptibility covaried with lower MMSE and MoCA scores, and was predominantly located in the right parietal cortex and lateral occipital cortex. In the AD group, cortical thickness was reduced in the left superior temporal gyrus, right frontal pole, fusiform gyus, and pars opercularis, and there were increases in susceptibility in the right frontal pole (AD: mean ± SD 0.034±0.007 ppm, 95% CI: 0.032-0.037 ppm; HC: 0.030±0.005 ppm, 95% CI: 0.028-0.032 ppm; P=0.016) and pars opercularis (AD: 0.020±0.003 ppm, 95% CI: 0.018-0.021 ppm; HC: 0.017±0.002 ppm, 95% CI: 0.017-0.018 ppm; P=0.002). Susceptibility was negatively correlated with cortical thickness in the right pars opercularis in the entire cohort (r=-0.521, P<0.001) and AD group (r=-0.510, P=0.005). Conclusions: Widespread cortical iron, as measured by QSM, accumulated in AD and iron deposition was associated with poor cognitive performance. Increased iron content was also associated with brain atrophy. Our study suggests QSM may be a useful imaging biomarker for monitoring the neurodegenerative progression of AD.

Water-Lubricated Aluminum Vanadate for Enhanced Rechargeable Magnesium Ion Storage.

Magnesium ion batteries (MIBs) have attracted much attention due to their low cost and high safety properties. However, the intense charge repulsion effect and sluggish diffusion dynamics of Mg2+ ions result in unsatisfactory electrochemical performance of conventional cathode materials in MIBs. This work reports water-lubricated aluminum vanadate (HAlVO) as high-performance cathode material for Mg2+ ions storage and investigates the capacity fade mechanism of water-free aluminum vanadate (AlVO). The charge density difference based on density functional theory calculation is performed to analyze the charge transfer process of water-lubricated/free aluminum vanadates (HAlVO/AlVO). The different charge transfer phenomena of two materials and the charge shielding effect of water molecule in HAlVO are revealed. Moreover, the single-phase structural evolution process and the Mg2+ ions storage mechanism of HAlVO are further investigated deeply by different in situ and ex situ characterization methods. This work proves that HAlVO is a potential candidate cathode material to satisfy the high-performance reversible Mg2+ ions storage, and the water-lubricated method is an effective strategy to improve the electrochemical performance of vanadium oxides cathode.

Five genes involved in circular RNA-associated competitive endogenous RNA network correlates with metastasis in papillary thyroid carcinoma.

This study aimed to identify potential circular RNA (circRNA), microRNA (miRNA) and mRNA biomarkers as well as their underlying regulatory mechanisms in papillary thyroid carcinoma (PTC). Three microarray datasets from the Gene Expression Omnibus database as well as expression data and clinical phenotype from The Cancer Genome Atlas (TCGA) were downloaded, followed by differential expression, functional enrichment, protein-protein interaction (PPI), and module analyses. The support vector machine (SVM)-recursive feature elimination (RFE) algorithm was used to screen the key circRNAs. Finally, the mRNA-miRNA-circRNA regulatory network and competitive endogenous RNA (ceRNA) network were constructed. The prognostic value and clinical correlations of key mRNAs were investigated using TCGA dataset, and their expression was validated using the UALCAN database. A total of 1039 mRNAs, 18 miRNAs and 137 circRNAs were differentially expressed in patients with PTC. A total of 37 key circRNAs were obtained using the SVM-RFE algorithm, whereas 46 key mRNAs were obtained from significant modules in the PPI network. A total of 11 circRNA-miRNA pairs and 40 miRNA-mRNA pairs were predicted. Based on these interaction pairs, 46 circRNA-miRNA-mRNA regulatory pairs were integrated, of which 8 regulatory pairs in line with the ceRNA hypothesis were obtained, including two circRNAs (circ_0004053 and circ_0028198), three miRNAs (miR-199a-5p, miR-199b-5p, and miR-7-5p), and five mRNAs, namely APOA2, CCL20, LPAR5, MFGE8, and TIMP1. Survival analysis showed that LPAR5 expression was associated with patient survival. APOA2 expression showed significant differences between metastatic and non-metastatic tumors, whereas CCL20, LPAR5, MFGE8 and TIMP1 showed significant differences between metastatic and non-metastatic lymph nodes. Overall, we identified several potential targets and regulatory mechanisms involved in PTC. APOA2, CCL20, LPAR5, MFGE8, and TIMP1 may be correlated with PTC metastasis.

Coupling HDAC4 with transcriptional factor MEF2D abrogates SPRY4-mediated suppression of ERK activation and elicits hepatocellular carcinoma drug resistance.

Hepatocellular carcinoma (HCC) lacks effective treatment, and the patients rapidly develop the acquired resistance to sorafenib with less defined mechanisms. Here, we demonstrate that transcriptional factor myocyte enhancer factor 2D (MEF2D) overexpression is detected in sorafenib-resistant HCC specimens and HCC cell lines and predicts poor prognosis of sorafenib-treated HCC patients. Mechanistically, MEF2D in complex with histone deacetylase HDAC4 directly binds to the SPRY4 promoter regions and suppresses the transcriptional expression of SPRY4, which is a negative regulator of MAPK/ERK signaling pathway. Inhibition of HDAC4 with its clinically used inhibitor induces SPRY4 expression and inhibition of ERK activity, resulting in sensitization of HCC cells to sorafenib-induced apoptosis and greatly improved inhibition of liver tumor growth in mice with sorafenib treatment. These findings highlight the critical role of coupling HDAC4 with MEF2D in activation of ERK by suppressing SPRY4 and underscore the great potential to improve HCC treatment by combined administration of sorafenib with HDAC4 inhibitors.

Novel aluminum vanadate as a cathode material for high-performance aqueous zinc-ion batteries.

Rechargeable aqueous zinc-ion batteries (AZIBs) have garnered widespread attention as a new large-scale energy storage candidate owing to their low cost and high theoretical capacity. Because of the unique divalent state of Zn2+and the existence of a strong electrostatic repulsion phenomenon, researchers are currently focusing on how to prepare high-performance cathode materials. In this study, we synthesized aluminum vanadate (AlV3O9) as a cathode material for AZIBs using a solvothermal method. Al3+acted as a pillar in the resultant structure and stabilized it. Furthermore, this large interlayer spacing enhanced the ion diffusion coefficient and accelerated the ion transport process. Because of these advantages, the AlV3O9(AVO) cathode exhibited excellent electrochemical performance, including a high capacity of 421.0 mA h g-1at 0.1 A g-1and a stable rate capability of 348.2 mA h g-1at 1 A g-1. Moreover, it exhibited a specific capacity of 202 mA h g-1even at a high current density of 3 A g-1(the capacity retention rate reached 84.38% after 1600 cycles). The prepared ZIBs presented a high power density of 366.6 W kg-1at an energy density of 286 W h kg-1. These extraordinary results indicate the great application potential of AVO as a cathode material for AZIBs.

Clinical evaluation of preoperative embolization, vertebroplasty, and decompression in the treatment of aggressive vertebral hemangiomas.

OBJECTIVE: To evaluate the efficacy and safety of preoperative embolization and vertebroplasty in the treatment of aggressive hemangioma. METHODS: A retrospective clinical review of patients diagnosed with aggressive vertebral hemangiomas was conducted. All the patients were assigned to three groups according to the treatment strategies: patients in Group A underwent embolization and decompression with internal fixation, patients in Group B underwent vertebroplasty and decompression with internal fixation, patients in Group C received all three treatments. Clinical indexes were compared within three groups. RESULTS: There were 16 patients received embolization and decompression (Group A), 19 patients underwent decompression with vertebroplasty (Group B) and 16 patients in Group C. The operative duration of patients in group A (198.33 ± 38.43 min) were less than another two groups (p = 0.001). The intraoperative blood loss of patients in group C was 713.33 ± 165.13 mL, which was significantly less than group A and group B (p = 0.045). Patients in group C exhibited the lowest volume of drainage on POD 1 (178.33 ± 66.76 mL), which showed significant difference compared with group A (368.33 ± 191.15 mL, p = 0.01). There was no significant difference of preoperative and postoperative VAS and JOA score among three groups, as well as drainage on POD 2, total volume and hospital duration. CONCLUSION: Both embolization and vertebroplasty are efficient and safe measures to reduce blood loss in the surgical treatment of aggressive vertebral hemangiomas, combination of all three methods is also competent.

The Phosphoenolpyruvate Carboxykinase Is a Key Metabolic Enzyme and Critical Virulence Factor of Leishmania major.

There is currently no effective vaccine against leishmaniasis because of the lack of sufficient knowledge about the Ags that stimulate host-protective and long-lasting T cell-mediated immunity. We previously identified Leishmania phosphoenolpyruvate carboxykinase (PEPCK, a gluconeogenic enzyme) as an immunodominant Ag that is expressed by both the insect (promastigote) and mammalian (amastigote) stages of the parasite. In this study, we investigated the role of PEPCK in metabolism, virulence, and immunopathogenicity of Leishmania major We show that targeted loss of PEPCK results in impaired proliferation of L. major in axenic culture and bone marrow-derived macrophages. Furthermore, the deficiency of PEPCK results in highly attenuated pathology in vivo. BALB/c mice infected with PEPCK-deficient parasites failed to develop any cutaneous lesions despite harboring parasites at the cutaneous site of infection. This was associated with a dramatic reduction in the frequency of cytokine (IFN-γ, IL-4, and IL-10)-producing CD4+ T cells in spleens and lymph nodes draining the infection site. Cells from mice infected with PEPCK-deficient parasites also produced significantly low levels of these cytokines into the culture supernatant following in vitro restimulation with soluble Leishmania Ag. PEPCK-deficient parasites exhibited significantly greater extracellular acidification rate, increased proton leak, and decreased ATP-coupling efficiency and oxygen consumption rates in comparison with their wild-type and addback counterparts. Taken together, these results show that PEPCK is a critical metabolic enzyme for Leishmania, and its deletion results in altered metabolic activity and attenuation of virulence.

Sensing Host Arginine Is Essential for Leishmania Parasites' Intracellular Development.

Arginine homeostasis in lysosomes is critical for the growth and metabolism of mammalian cells. Phagolysosomes of macrophages are the niche where the parasitic protozoan Leishmania resides and causes human leishmaniasis. During infection, parasites encounter arginine deprivation, which is monitored by a sensor on the parasite cell surface. The sensor promptly activates a mitogen-activated protein kinase 2 (MAPK2)-mediated arginine deprivation response (ADR) pathway, resulting in upregulating the abundance and activity of the Leishmania arginine transporter (AAP3). Significantly, the ADR is also activated during macrophage infection, implying that arginine levels within the host phagolysosome are limiting for growth. We hypothesize that ADR-mediated upregulation of AAP3 activity is necessary to withstand arginine starvation, suggesting that the ADR is essential for parasite intracellular development. CRISPR/Cas9-mediated disruption of the AAP3 locus yielded mutants that retain a basal level of arginine transport but lack the ability to respond to arginine starvation. While these mutants grow normally in culture, they were impaired in their ability to develop inside THP-1 macrophages and were ∼70 to 80% less infective in BALB/c mice. Hence, inside the host macrophage, Leishmania must overcome the arginine "hunger games" by upregulating the transport of arginine via the ADR. We show that the ability to monitor and respond to changes in host metabolite levels is essential for pathogenesis.IMPORTANCE In this study, we report that the ability of the human pathogen Leishmania to sense and monitor the lack of arginine in the phagolysosome of the host macrophage is essential for disease development. Phagolysosomes of macrophages are the niche where Leishmania resides and causes human leishmaniasis. During infection, the arginine concentration in the phagolysosome decreases as part of the host innate immune response. An arginine sensor on the Leishmania cell surface activates an arginine deprivation response pathway that upregulates the expression of a parasite arginine transporter (AAP3). Here, we use CRISPR/Cas9-mediated disruption of the AAP3 locus to show that this response enables Leishmania parasites to successfully compete with the host macrophage in the "hunger games" for arginine.

Cytokines in abdominal exudate and serum predict small bowel obstruction following appendectomy.

BACKGROUND: This study aimed to investigate the value of inflammatory markers for the prediction of small bowel obstruction (SBO) following appendectomy. METHODS: We included cases of acute appendicitis that underwent laparoscopic appendectomy (LA) in the Qingdao Municipal Hospital between January 2017 and January 2019. The cases were divided into an SBO group and a non-SBO group depending on whether patients had or did not have SBO, and patients were followed up for at least 1 year. The levels of interleukin (IL)-1ß, IL-6 and tumour necrosis factor-alpha (TNF-α) in abdominal exudate and venous blood were examined using enzyme-linked immunosorbent assay. RESULTS: After 1 year of follow-up, there were 985 cases in the non-SBO group and 16 cases in the SBO group. The levels of IL-1ß, IL-6 and TNF-α in abdominal exudate on post-operative day 1 in the SBO group were 172.5 ± 14.7, 2167.3 ± 372.1 and 253.9 ± 12.9 pg/mL, respectively, which were significantly higher than that in the non-SBO group. The serum levels of IL-1ß, IL-6, TNF-α and C-reactive protein (CRP) in the SBO group were significantly higher than that in the non-SBO group before surgery. Post-operatively, the inflammatory markers above decreased significantly and became similar with time in both groups. The logistic regression showed that the levels of peritoneal IL-6, preoperative serum CRP and perforated appendicitis were significant risk factors of SBO. The specificity and sensitivity of peritoneal IL-6 were 0.81 and 0.921, respectively. CONCLUSION: The IL-1ß, IL-6, TNF-α and CRP in serum and abdominal exudate played an important role in SBO after LA. The peritoneal IL-6 was the most reliable prediction marker for SBO.

Attenuation of Oxidative Injury With Targeted Expression of NADPH Oxidase 2 Short Hairpin RNA Prevents Onset and Maintenance of Electrical Remodeling in the Canine Atrium: A Novel Gene Therapy Approach to Atrial Fibrillation.

BACKGROUND: Atrial fibrillation (AF) is the most common heart rhythm disorder in adults and a major cause of stroke. Unfortunately, current treatments of AF are suboptimal because they are not targeted to the molecular mechanisms underlying AF. Using a highly novel gene therapy approach in a canine, rapid atrial pacing model of AF, we demonstrate that NADPH oxidase 2 (NOX2) generated oxidative injury causes upregulation of a constitutively active form of acetylcholine-dependent K+ current (IKACh), called IKH; this is an important mechanism underlying not only the genesis, but also the perpetuation of electric remodeling in the intact, fibrillating atrium. METHODS: To understand the mechanism by which oxidative injury promotes the genesis and maintenance of AF, we performed targeted injection of NOX2 short hairpin RNA (followed by electroporation to facilitate gene delivery) in atria of healthy dogs followed by rapid atrial pacing. We used in vivo high-density electric mapping, isolation of atrial myocytes, whole-cell patch clamping, in vitro tachypacing of atrial myocytes, lucigenin chemiluminescence assay, immunoblotting, real-time polymerase chain reaction, immunohistochemistry, and Masson trichrome staining. RESULTS: First, we demonstrate that generation of oxidative injury in atrial myocytes is a frequency-dependent process, with rapid pacing in canine atrial myocytes inducing oxidative injury through the induction of NOX2 and the generation of mitochondrial reactive oxygen species. We show that oxidative injury likely contributes to electric remodeling in AF by upregulating IKACh by a mechanism involving frequency-dependent activation of PKCε (protein kinase C epsilon). The time to onset of nonsustained AF increased by >5-fold in NOX2 short hairpin RNA-treated dogs. Furthermore, animals treated with NOX2 short hairpin RNA did not develop sustained AF for up to 12 weeks. The electrophysiological mechanism underlying AF prevention was prolongation of atrial effective refractory periods, at least in part attributable to the attenuation of IKACh. Attenuated membrane translocation of PKCε appeared to be a likely molecular mechanism underlying this beneficial electrophysiological remodeling. CONCLUSIONS: NOX2 oxidative injury (1) underlies the onset, and the maintenance of electric remodeling in AF, as well, and (2) can be successfully prevented with a novel, gene-based approach. Future optimization of this approach may lead to a novel, mechanism-guided therapy for AF.

A second generation leishmanization vaccine with a markerless attenuated Leishmania major strain using CRISPR gene editing.

Leishmaniasis is a neglected tropical disease caused by Leishmania protozoa transmitted by infected sand flies. Vaccination through leishmanization with live Leishmania major has been used successfully but is no longer practiced because it resulted in occasional skin lesions. A second generation leishmanization is described here using a CRISPR genome edited L. major strain (LmCen-/-). Notably, LmCen-/- is a genetically engineered centrin gene knock-out mutant strain that is antibiotic resistant marker free and does not have detectable off-target mutations. Mice immunized with LmCen-/- have no visible lesions following challenge with L. major-infected sand flies, while non-immunized animals develop large and progressive lesions with a 2-log fold higher parasite burden. LmCen-/- immunization results in protection and an immune response comparable to leishmanization. LmCen-/- is safe since it is unable to cause disease in immunocompromised mice, induces robust host protection against vector sand fly challenge and because it is marker free, can be advanced to human vaccine trials.

Forkhead box protein O1 (FoxO1) regulates hepatic serine protease inhibitor B1 (serpinB1) expression in a non-cell-autonomous fashion.

FoxO proteins are major targets of insulin action, and FoxO1 mediates the effects of insulin on hepatic glucose metabolism. We reported previously that serpinB1 is a liver-secreted factor (hepatokine) that promotes adaptive ß-cell proliferation in response to insulin resistance in the liver-specific insulin receptor knockout (LIRKO) mouse. Here we report that FoxO1 plays a critical role in promoting serpinB1 expression in hepatic insulin resistance in a non-cell-autonomous manner. Mice lacking both the insulin receptor and FoxO1 (LIRFKO) exhibit reduced ß-cell mass compared with LIRKO mice because of attenuation of ß-cell proliferation. Although hepatic expression of serpinB1 mRNA and protein levels was increased in LIRKO mice, both the mRNA and protein levels returned to control levels in LIRFKO mice. Furthermore, liver-specific expression of constitutively active FoxO1 in transgenic mice induced an increase in hepatic serpinB1 mRNA and protein levels in refed mice. Conversely, serpinB1 mRNA and protein levels were reduced in mice lacking FoxO proteins in the liver. ChIP studies demonstrated that FoxO1 binds to three distinct sites located ∼9 kb upstream of the serpinb1 gene in primary mouse hepatocytes and that this binding is enhanced in hepatocytes from LIRKO mice. However, adenoviral expression of WT or constitutively active FoxO1 and insulin treatment are sufficient to regulate other FoxO1 target genes (IGFBP-1 and PEPCK) but not serpinB1 expression in mouse primary hepatocytes. These results indicate that liver FoxO1 promotes serpinB1 expression in hepatic insulin resistance and that non-cell-autonomous factors contribute to FoxO1-dependent effects on serpinB1 expression in the liver.

Identification of key genes and miRNAs markers of papillary thyroid cancer.

OBJECTIVE: In this study, crucial genes and microRNAs (miRNAs) associated with the progression, staging, and prognosis of papillary thyroid cancer (PTC) were identified. METHODS: Four PTC datasets, including our own mRNA-sequencing (mRNA-seq) dataset and three public datasets downloaded from Gene Expression Omnibus and The Cancer Genome Atlas, were used to analyze differentially expressed genes (DEGs) and miRNAs (DEMs) between PTC tumor tissues and paired normal tissues (control). Gene ontology (GO) terms and pathways associated with these DEGs were identified, and protein-protein interactions (PPIs) were analyzed. Additionally, an miRNA-mRNA regulatory network was constructed and the functions of DEMs were explored. Finally, miRNAs/mRNAs associated with tumor staging and prognosis were identified. The expression levels of several key genes and miRNAs were validated by qRT-PCR. RESULTS: Numerous DEGs and DEMs were identified between tumor and control groups in four datasets. The DEGs were significantly enriched in cell adhesion and cancer-related GO terms and pathways. In the constructed PPI network, ITGA2, FN1, ICAM1, TIMP1 and CDH2 were hub proteins. In the miRNA-mRNA negative regulatory networks, miR-204-5p regulated the largest number of target genes, such as TNFRSF12A. miR-146b, miR-204, miR-7-2, and FN1 were associated with tumor stage in PTC, and TNFRSF12A and CLDN1 were related to prognosis. CONCLUSIONS: Our results suggested the important roles of ITGA2, FN1, ICAM1, TIMP1 and CDH2 in the progression of PTC. miR-204-5p, miR-7-2, and miR-146b are potential biomarkers for PTC staging and FN1, CLDN1, and TNFRSF12A may serve as markers of prognosis in PTC.

Advanced Whole-Genome Sequencing and Analysis of Fetal Genomes from Amniotic Fluid.

BACKGROUND: Amniocentesis is a common procedure, the primary purpose of which is to collect cells from the fetus to allow testing for abnormal chromosomes, altered chromosomal copy number, or a small number of genes that have small single- to multibase defects. Here we demonstrate the feasibility of generating an accurate whole-genome sequence of a fetus from either the cellular or cell-free DNA (cfDNA) of an amniotic sample. METHODS: cfDNA and DNA isolated from the cell pellet of 31 amniocenteses were sequenced to approximately 50× genome coverage by use of the Complete Genomics nanoarray platform. In a subset of the samples, long fragment read libraries were generated from DNA isolated from cells and sequenced to approximately 100× genome coverage. RESULTS: Concordance of variant calls between the 2 DNA sources and with parental libraries was >96%. Two fetal genomes were found to harbor potentially detrimental variants in chromodomain helicase DNA binding protein 8 (CHD8) and LDL receptor-related protein 1 (LRP1), variations of which have been associated with autism spectrum disorder and keratosis pilaris atrophicans, respectively. We also discovered drug sensitivities and carrier information of fetuses for a variety of diseases. CONCLUSIONS: We were able to elucidate the complete genome sequence of 31 fetuses from amniotic fluid and demonstrate that the cfDNA or DNA from the cell pellet can be analyzed with little difference in quality. We believe that current technologies could analyze this material in a highly accurate and complete manner and that analyses like these should be considered for addition to current amniocentesis procedures.

Germline and somatic variant identification using BGISEQ-500 and HiSeq X Ten whole genome sequencing.

Technological innovation and increased affordability have contributed to the widespread adoption of genome sequencing technologies in biomedical research. In particular large cancer research consortia have embraced next generation sequencing, and have used the technology to define the somatic mutation landscape of multiple cancer types. These studies have primarily utilised the Illumina HiSeq platforms. In this study we performed whole genome sequencing of three malignant pleural mesothelioma and matched normal samples using a new platform, the BGISEQ-500, and compared the results obtained with Illumina HiSeq X Ten. Germline and somatic, single nucleotide variants and small insertions or deletions were independently identified from data aligned human genome reference. The BGISEQ-500 and HiSeq X Ten platforms showed high concordance for germline calls with genotypes from SNP arrays (>99%). The germline and somatic single nucleotide variants identified in both sequencing platforms were highly concordant (86% and 72% respectively). These results indicate the potential applicability of the BGISEQ-500 platform for the identification of somatic and germline single nucleotide variants by whole genome sequencing. The BGISEQ-500 datasets described here represent the first publicly-available cancer genome sequencing performed using this platform.