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Yuyan Sun,1 Bing Lu,1 Yuanwen Hu,2 Yingqi Lv,3 Shao Zhong1 1Department of Endocrinology, Gusu School, Nanjing Medical University, The First People’s Hospital of Kunshan, Kunshan, 215300, People’s Republic of China; 2Department of Gastroenterology, The First People’s Hospital of Kunshan, Kunshan, 215300, People’s Republic of China; 3Division of Endocrinology, Zhongda Hospital, School of Medicine, Southeast University, Nanjing, 210009, People’s Republic of ChinaCorrespondence: Shao Zhong, Department of Endocrinology, Gusu School, Nanjing Medical University, the First People’s Hospital of Kunshan, Kunshan, 215300, People’s Republic of China, Tel +86 13328056828, Email drzhong@163.comAbstract: In recent years, pancreatogenic diabetes mellitus has garnered significant attention due to its high incidence, complications, and mortality rates. Glycemic variability (GV) can increase the risk of pancreatogenic diabetes mellitus and its associated complications; however, the precise mechanism remains unclear. The effective control of GV is crucial for preventing the onset of pancreatic diabetes mellitus and improving prognosis. Both diet and antidiabetic medications have substantial effects on GV. However, many patients are prescribed suboptimal or even harmful drugs. Therefore, to provide a comprehensive treatment basis for clinicians to prevent and treat pancreatogenic diabetes mellitus, this study aimed to elucidate the relationship between GV and pancreatogenic diabetes mellitus; investigate the potential mechanisms (such as oxidative stress, inflammatory response, insulin resistance, and lipid metabolism disorders); provide lifestyle guidance; and recommend drug selections to reduce the GV in patients with pancreatogenic diabetes mellitus. Keywords: pancreatogenic diabetes, glucose fluctuation, glycemic variability, therapy, antihyperglycemic drug

Luxun Yang,1,* Haiying Ran,2,* Yaru Yin,1 Jing Liu,1 Binghui Lu,1 Xi Ran,3 Shenglin Luo,1 Weidong Wang,4 Zhangyou Yang,5 Rong Li1 1Institute of Combined Injury, National Key Laboratory of Trauma and Chemical Poisoning, Army Key Laboratory of Nanomedicine, Department of Military Preventive Medicine, Army Medical University, Chongqing, 400038, People’s Republic of China; 2Biomedical Analysis Center, Army Medical University, Chongqing, 400038, People’s Republic of China; 3Department of Medical Laboratory, Xinqiao Hospital, Army Medical University, Chongqing, 400037, People’s Republic of China; 4Department of Radiation Oncology, Sichuan Cancer Hospital, Chengdu, 610041, People’s Republic of China; 5Chongqing Key Laboratory for Pharmaceutical Metabolism Research, Chongqing Pharmacodynamic Evaluation Engineering Technology Research Center, College of Pharmacy, Chongqing Medical University, Chongqing, 400016, People’s Republic of China*These authors contributed equally to this workCorrespondence: Zhangyou Yang, Chongqing Medical University, No. 1, Yixueyuan Road, Chongqing, 400016, People’s Republic of China, Email yangzhangyou@cqmu.edu.cn Rong Li, Army Medical University, No. 30, Gaotanyan Street, Chongqing, 400038, People’s Republic of China, Tel +86-13883996627, Fax +8623-68753127, Email lrong361@126.comPurpose: Mitochondrial oxidative stress is an important factor in cell apoptosis. Cerium oxide nanomaterials show great potential for scavenging free radicals and simulating superoxide dismutase (SOD) and catalase (CAT) activities. To solve the problem of poor targeting of cerium oxide nanomaterials, we designed albumin-cerium oxide nanoclusters (TPP-PCNLs) that target the modification of mitochondria with triphenyl phosphate (TPP). TPP-PCNLs are expected to simulate the activity of superoxide dismutase, continuously remove reactive oxygen species, and play a lasting role in radiation protection.Methods: First, cerium dioxide nanoclusters (CNLs), polyethylene glycol cerium dioxide nanoclusters (PCNLs), and TPP-PCNLs were characterized in terms of their morphology and size, ultraviolet spectrum, dispersion stability and cellular uptake, and colocalization Subsequently, the anti-radiation effects of TPP-PCNLs were investigated using in vitro and in vivo experiments including cell viability, apoptosis, comet assays, histopathology, and dose reduction factor (DRF).Results: TPP-PCNLs exhibited good stability and biocompatibility. In vitro experiments indicated that TPP-PCNLs could not only target mitochondria excellently but also regulate reactive oxygen species (ROS)levels in whole cells. More importantly, TPP-PCNLs improved the integrity and functionality of mitochondria in irradiated L-02 cells, thereby indirectly eliminating the continuous damage to nuclear DNA caused by mitochondrial oxidative stress. TPP-PCNLs are mainly targeted to the liver, spleen, and other extramedullary hematopoietic organs with a radiation dose reduction factor of 1.30. In vivo experiments showed that TPP-PCNLs effectively improved the survival rate, weight change, hematopoietic function of irradiated animals. Western blot experiments have confirmed that TPP-PCNLs play a role in radiation protection by regulating the mitochondrial apoptotic pathway.Conclusion: TPP-PCNLs play a radiologically protective role by targeting extramedullary hematopoietic organ-liver cells and mitochondria to continuously clear ROS.Keywords: radioprotective effect, ceria nanoclusters, mitochondria targeting, oxidative stress, superoxide dismutase

Abstract Background Survival prediction using high-dimensional molecular data is a hot topic in the field of genomics and precision medicine, especially for cancer studies. Considering that carcinogenesis has a pathway-based pathogenesis, developing models using such group structures is a closer mimic of disease progression and prognosis. Many approaches can be used to integrate group information; however, most of them are single-model methods, which may account for unstable prediction. Methods We introduced a novel survival stacking method that modeled using group structure information to improve the robustness of cancer survival prediction in the context of high-dimensional omics data. With a super learner, survival stacking combines the prediction from multiple sub-models that are independently trained using the features in pre-grouped biological pathways. In addition to a non-negative linear combination of sub-models, we extended the super learner to non-negative Bayesian hierarchical generalized linear model and artificial neural network. We compared the proposed modeling strategy with the widely used survival penalized method Lasso Cox and several group penalized methods, e.g., group Lasso Cox, via simulation study and real-world data application. Results The proposed survival stacking method showed superior and robust performance in terms of discrimination compared with single-model methods in case of high-noise simulated data and real-world data. The non-negative Bayesian stacking method can identify important biological signal pathways and genes that are associated with the prognosis of cancer. Conclusions This study proposed a novel survival stacking strategy incorporating biological group information into the cancer prognosis models. Additionally, this study extended the super learner to non-negative Bayesian model and ANN, enriching the combination of sub-models. The proposed Bayesian stacking strategy exhibited favorable properties in the prediction and interpretation of complex survival data, which may aid in discovering cancer targets.

Lung cancer is a leading cause of cancer death worldwide, with high incidence and poor survival rates. Cold atmospheric plasma (CAP) technology has emerged as a promising therapeutic approach for cancer treatment, inducing oxidative stress in malignant tissues without causing thermal damage. However, the role of CAP in regulating lung cancer cell ferroptosis remains unclear. Here, we observed that CAP effectively suppressed the growth and migration abilities of lung cancer cells, with significantly increased ferroptotic cell death, lipid peroxidation, and decreased mitochondrial membrane potential. Mechanistically, CAP regulates SLC7A11-mediated cell ferroptosis by modulating HOXB9. SLC7A11, a potent ferroptosis suppressor, was markedly reduced by HOXB9 knockdown, while it was enhanced by overexpressing HOXB9. The luciferase and ChIP assays confirmed that HOXB9 can directly target SLC7A11 and regulate its gene transcription. Additionally, CAP enhanced the acetylation modification level of HOXB9 by promoting its interaction with acetyltransferase p300/CBP-associated factor (PCAF). Acetylated HOXB9 affects its protein ubiquitination modification level, which in turn affects its protein stability. Notably, the upregulation of SLC7A11 and HOXB9 mitigated the suppressive effects of CAP on ferroptosis status, cell proliferation, invasion, and migration in lung cancer cells. Furthermore, animal models have also confirmed that CAP can inhibit the progression of lung cancer in vivo. Overall, this study highlights the significance of the downregulation of the HOXB9/SLC7A11 axis by CAP treatment in inhibiting lung cancer, offering novel insights into the potential mechanisms and therapeutic strategies of CAP for lung cancer.

Yixiu Zhong,1 Youfan Zhang,2 Beibei Lu,1 Zhenjun Deng,1 Zhiwen Zhang,2 Qi Wang,2 Jianglin Zhang1 1Department of Dermatology, Shenzhen People’s Hospital (The Second Clinical Medical College, Jinan University; The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen, Guangdong, People’s Republic of China; 2Department of Dermatology, Nanfang Hospital, Southern Medical University, Guangzhou, People’s Republic of ChinaCorrespondence: Yixiu Zhong; Jianglin Zhang, Email 846783501@qq.com; zhang.jianglin@szhospital.comAbstract: Hypertrophic scars and keloids are common fibroproliferative diseases following injury. Patients with pathologic scars suffer from impaired quality of life and psychological health due to appearance disfiguration, itch, pain, and movement disorders. Recently, the advancement of hydrogels in biomedical fields has brought a variety of novel materials, methods and therapeutic targets for treating hypertrophic scars and keloids, which exhibit broad prospects. This review has summarized current research on hydrogels and loaded components used in preventing and treating hypertrophic scars and keloids. These hydrogels attenuate keloid and hypertrophic scar formation and progression by loading organic chemicals, drugs, or bioactive molecules (such as growth factors, genes, proteins/peptides, and stem cells/exosomes). Among them, smart hydrogels (a very promising method for loading many types of bioactive components) are currently favoured by researchers. In addition, combining hydrogels and current therapy (such as laser or radiation therapy, etc.) could improve the treatment of hypertrophic scars and keloids. Then, the difficulties and limitations of the current research and possible suggestions for improvement are listed. Moreover, we also propose novel strategies for facilitating the construction of target multifunctional hydrogels in the future. Keywords: hydrogel, wound healing, hypertrophic scar, keloid

Purpose: Previous studies have confirmed that Achilles tendon occurs Achilles thickening after repair surgery of the rupture. Although this mechanism has been elucidated in the laboratory, there are few reports on its impact on clinical function. We designed a retrospective study to investigate the Achilles thickening after Achilles tendon rupture repair and its correlation between the elasticity and postoperative function. Methods: In this retrospective analysis, patients who underwent surgical treatment for acute Achilles tendon rupture from April 2016 to April 2020 were included. All the patients were regularly followed up at 3 months, 1 year, and 2 years after surgery. American Orthopaedic Foot Ankle Surgeon (AOFAS) scale and Leppilahti score were used to evaluate functional outcomes. Achilles elasticity was measured by ultrasound shear wave of elasticity. Achilles thickening was calculated as maximal transverse and longitudinal diameter in cross-sectional plane of magnetic resonance scan. Sample t-tests was used for different follow-up periods. Correlation between Achilles thickening and other factors were analyzed using Pearson's method. p 0.05) in every follow-up period. Conclusion: Achilles tendon thickens after surgery in the 1st year, but begins to gradually return to thinning about 2 years after surgery. There was no significant correlation between the increase and decrease of thickening and the patients' clinical function scores, Achilles elasticity, and bilateral ankle dorsiflexion difference.

Circulating cfRNA in plasma has emerged as a fascinating area of research with potential applications in disease diagnosis, monitoring, and personalized medicine. Circulating RNA sequencing technology allows for the non-invasive collection of important information about the expression of target genes, eliminating the need for biopsies. This comprehensive review aims to provide a detailed overview of the current knowledge and advancements in the study of plasma cfRNA, focusing on its diverse landscape and biological functions, detection methods, its diagnostic and prognostic potential in various diseases, challenges, and future perspectives.

Zhongbiao Nie,1 Xianmei Cui,2 Ran Zhang,3 Zhihong Li,1 Bin Lu,4 Suxian Li,1 Tao Cao,2 Ping Zhuang4 1Pharmaceutical Department, Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Tongji Shanxi Hospital, Third Hospital of Shanxi Medical University, Taiyuan, 030012, People’s Republic of China; 2Obstetrics Department, Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Tongji Shanxi Hospital, Third Hospital of Shanxi Medical University, Taiyuan, 030012, People’s Republic of China; 3Nephrology Department, Affiliated Hospital of Shanxi University of Traditional Chinese Medicine, Taiyuan, 030036, People’s Republic of China; 4Anesthesiology Department, Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Tongji Shanxi Hospital, Third Hospital of Shanxi Medical University, Taiyuan, 030012, People’s Republic of ChinaCorrespondence: Ping Zhuang, Anesthesiology Department, Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Tongji Shanxi Hospital, Third Hospital of Shanxi Medical University, Taiyuan, 030012, People’s Republic of China, Tel +86-13700503507, Email 13700503507@163.comPurpose: To investigate the effectiveness of sufentanil patient-controlled intravenous analgesia pump (PCIA) and background infusion in patients of post-cesarean analgesia.Patients and Methods: This trial compared two groups of women undergoing cesarean section and receiving PCIA: no background infusion group (n=30), 6-min lockout time, and background infusion group (n=30), 2 mL/h infusion, 10-min lockout time. Both groups with 2 μg/kg sufentanil was diluted to 100 mL with normal saline. VAS scores at rest at 36 h was the primary endpoint. The secondary endpoints were the VAS scores at rest at 6, 12, and 24 h, the total amount of sufentanil consumed, the Ramsay sedation score (RSS) assessed at the same time points, postpartum bleeding within 24 h, the injection/attempt (I/A) ratio, BP and HR, PONV, side effects of sufentanil.Results: Compared with the no background infusion group, the background infusion group showed lower VAS pain scores at 6, 12, and 24 h (P< 0.01), but no differences at 36 h (95% CI = − 0.5– 0.8. P> 0.05). Attempts, injections, and total sufentanil consumption were significantly different between the two groups (P< 0.001), but without difference in I/A. Bleeding was less in the background infusion group at 1 h (P=0.03). The minimal respiration rates were not significantly different between groups.Conclusion: Background infusion increased the total consumption of sufentanil within 36 h after cesarean section. Although it did not reduce uterine contraction pain and wound pain at 36 h, it significantly reduced the pain at 6, 12, and 24 h after cesarean section. It improved patient satisfaction and reduced the amount of bleeding after 1 h. Importantly, it did not increase the incidence of hypertension, PONV and respiratory depression.Keywords: background infusion, patient-controlled intravenous analgesia, PCIA, sufentanil

Xiang Wang,1,2 Xin-Shuang Ren,3 Yun-Qiang An,3 Zhi-Hui Hou,3 Yi-Tong Yu,3 Bin Lu,3,&ast; Fang Wang1,2,&ast; 1Department of Cardiology, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, 100730, People’s Republic of China; 2Graduate School of Peking Union Medical College, Chinese Academy of Medical Science, Beijing, 100730, People’s Republic of China; 3Department of Radiology, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100037, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Fang WangDepartment of Cardiology, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, No. 1 DaHua Road, Dongcheng District, Beijing, 100730, People’s Republic of ChinaTel +86 10 85132266Email fw_wangdr@163.comBin LuDepartment of Radiology, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 167 Bei-Li-Shi Street, Xicheng District, Beijing, 100037, People’s Republic of ChinaTel +86 10 88398700Email binlu_bl09@163.comBackground: A limitation associated with coronary computed tomography angiography (CCTA) is the lack of a normal reference value for aortic root dimensions and the uncertainty of the influence of age and gender on these dimensions. The purpose of the present study was to identify the normal values and variations of aortic root dimensions in healthy individuals and investigate how gender and age affect aortic root size.Methods: A total of 1286 healthy yellow population (52.7 ± 11.0 years, 634 male) who underwent CCTA were retrospectively included in the present study. Male and female patients were divided into seven groups according to age (< 30 years old, 30– 39, 40– 49, 50– 59, 60– 69, 70– 79, ≥ 80 years old). In these age groups, we measured and compared the parameters of the aortic root.Results: After body surface area (BSA) correction, the aortic root parameters of females were found to be greater than those of males in the 40– 49 age group (P< 0.05). There were no significant differences in aortic root parameters between genders in other age groups, except for the diameter of the ascending aorta, which was greater in females (P< 0.05). In males, age was positively correlated with aortic root parameters (P< 0.05), except for the annulus short diameter and LVOT short diameter. In females, age was positively correlated with aortic root parameters (P< 0.05), except for the left coronary ostia height and the LVOT short diameter.Conclusion: Aortic root dimensions are affected by age and gender. After BSA correction, females show larger aortic root dimensions than males, and aortic root diameters increase with age.Keywords: aortic root, BSA, CCTA, age, gender

Ping Zhang,1 Julie Marill,1 Audrey Darmon,1 Naeemunnisa Mohamed Anesary,1 Bo Lu,2 Sébastien Paris1 1Nanobiotix, Paris, France; 2Department of Radiation Oncology, Thomas Jefferson University, Philadelphia, PA, 19107, USACorrespondence: Sébastien ParisNanobiotix, 60 rue de Wattignies, Paris, 75012, FranceTel +33 1 75 44 72 33Fax +33 1 40 26 04 44Email sebastien.paris@nanobiotix.comPurpose: The side effects of radiotherapy induced on healthy tissue limit its use. To overcome this issue and fully exploit the potential of radiotherapy to treat cancers, the first-in-class radioenhancer NBTXR3 (functionalized hafnium oxide nanoparticles) has been designed to amplify the effects of radiotherapy.Patients and Methods: Thanks to its physical mode of action, NBTXR3 has the potential to be used to treat any type of solid tumor. Here we demonstrate that NBTXR3 can be used to treat a wide variety of solid cancers. For this, we evaluated different parameters on a large panel of human cancer models, such as nanoparticle endocytosis, in vitro cell death induction, dispersion, and retention of NBTXR3 in the tumor tissue and tumor growth control.Results: Whatever the model considered, we show that NBTXR3 was internalized by cancer cells and persisted within the tumors throughout radiotherapy treatment. NBTXR3 activated by radiotherapy was also more effective in destroying cancer cells and in controlling tumor growth than radiotherapy alone. Beyond the effects of NBTXR3 as single agent, we show that the antitumor efficacy of cisplatin-based chemoradiotherapy treatment was improved when combined with NBTXR3.Conclusion: These data support that NBTXR3 could be universally used to treat solid cancers when radiotherapy is indicated, opening promising new therapeutic perspectives of treatment for the benefit of many patients.Keywords: radiotherapy, radioenhancer, nanoparticle, NBTXR3, cancer

Jing Liu,1,* Yaru Yin,1,* Luxun Yang,1 Binghui Lu,1 Zhangyou Yang,2 Weidong Wang,3 Rong Li1 1Institute of Combined Injury, State Key Laboratory of Trauma, Burns and Combined Injury, Military Key Laboratory of Nanomedicine, Department of Military Preventive Medicine, Army Medical University, Chongqing, 400038, People’s Republic of China; 2Department of Pharmacy, Chongqing Medical University, Chongqing, 400010, People’s Republic of China; 3Department of Radiation Oncology, Sichuan Cancer Hospital, Chengdu, 610041, People’s Republic of China*These authors contributed equally to this workCorrespondence: Weidong WangSichuan Cancer Hospital, Renmin South Road, Chengdu, 610041, Sichuan, People’s Republic of ChinaEmail wwdwyl@sina.comRong LiArmy Medical University, No 30, Gaotanyan St., Chongqing, 400038, People’s Republic of ChinaTel +86-13883996627Fax +86023-68753127Email lrong361@126.comPurpose: The near-infrared fluorescent dye indocyanine green (ICG) has shown great potential in the photodynamic therapy (PDT) and photothermal therapy (PTT) of cancer. However, its disadvantages of instability in aqueous solution, short half-life, and non-targeting accumulation limit the effectiveness of ICG PDT/PTT. To overcome the disadvantages of ICG in tumor treatment, we designed PEGylated-human serum albumin (PHSA)-ICG-TAT. In this nanoparticle, PEG4000, the HSA package, and nuclear targeting peptide TAT (human immunodeficiency virus 1 [HIV-1]-transactivator protein) were used to improve the water solubility of ICG, prolong the life span of ICG in vivo, and target the nuclei of tumor cells, respectively.Methods: The PHSA-ICG-TAT was characterized in terms of morphology and size, ultraviolet spectrum, dispersion stability, singlet oxygen and cellular uptake, and colocalization using transmission electron microscopy and dynamic light scattering, and fluorescence assay, respectively. Subsequently, the anti-tumor effect of PHSA-ICG-TAT was investigated via in vitro and in vivo experiments, including cell viability, apoptosis, comet assays, histopathology, and inhibition curves.Results: The designed ICG-loaded nanoparticle had a higher cell uptake rate and stronger PDT/PTT effect than free ICG. The metabolism of PHSA-ICG-TAT in normal mice revealed that there was no perceptible toxicity. In vivo imaging of mice showed that PHSA-ICG-TAT had a good targeting effect on tumors. PHSA-ICG-TAT was used for the phototherapy of tumors, and significantly suppressed the tumor growth. The tumor tissue sections showed that the cell gap and morphology of the tumor tissue had been obviously altered after treatment with PHSA-ICG-TAT.Conclusion: These results indicate that the PHSA-ICG-TAT had a significant therapeutic effect against tumors.Keywords: indocyanine green, human serum albumin, phototherapy, nuclear targeting

Jing-Yi Ye,1 Zhong-Yun Chen,1 Chuan-Li Huang,1 Bei Huang,2 Yu-Rong Zheng,2 Ying-Feng Zhang,1 Ban-Yi Lu,2 Lin He,2 Chang-Shun Liu3 ,* Xiao-Ying Long1,4, * 1School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, People’s Republic of China; 2School of Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou 510006, People’s Republic of China; 3School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, 510515, People’s Republic of China; 4Guangdong Engineering & Technology Research Center of Topical Precise Drug Delivery System, Guangdong Pharmaceutical University, Guangzhou 510006, People’s Republic of China*These authors contributed equally to this workCorrespondence: Chang-Shun LiuSouthern Medical University, No. 1023-1063 of Shatai South Road, Guangzhou, Guangdong 510515, People’s Republic of ChinaTel +86 13430301554Email lcshun1225@163.comXiao-Ying LongGuangdong Pharmaceutical University, No. 280 of Waihuan East Road, Guangzhou, Guangdong 510006, People’s Republic of ChinaTel +86 13798171092Fax +86 2039352174Email longxy3156@163.comObjective: A non-lipolysis nanoemulsion (NNE) was designed to reduce the first-pass metabolism of raloxifene (RAL) by intestinal UDP-glucuronosyltransferases (UGTs) for increasing the oral absorption of RAL, coupled with in vitro and in vivo studies.Methods: In vitro stability of NNE was evaluated by lipolysis and the UGT metabolism system. The oral bioavailability of NNE was studied in rats and pigs. Finally, the absorption mechanisms of NNE were investigated by in situ single-pass intestinal perfusion (SPIP) in rats, Madin-Darby canine kidney (MDCK) cells model, and lymphatic blocking model.Results: The pre-NNE consisted of isopropyl palmitate, linoleic acid, Cremophor RH40, and ethanol in a weight ratio of 3.33:1.67:3:2. Compared to lipolysis nanoemulsion of RAL (RAL-LNE), the RAL-NNE was more stable in in vitro gastrointestinal buffers, lipolysis, and UGT metabolism system (p < 0.05). The oral bioavailability was significantly improved by the NNE (203.30%) and the LNE (205.89%) relative to the suspension group in rats. However, 541.28% relative bioavailability was achieved in pigs after oral NNE intake compared to the suspension and had two-fold greater bioavailability than the LNE (p < 0.05). The RAL-NNE was mainly absorbed in the jejunum and had high permeability at the intestine of rats. The results of both SPIP and MDCK cell models demonstrated that the RAL-NNE was absorbed via endocytosis mediated by caveolin and clathrin. The other absorption route, the lymphatic transport (cycloheximide as blocking agent), was significantly improved by the NNE compared with the LNE (p < 0.05).Conclusion: A NNE was successfully developed to reduce the first-pass metabolism of RAL in the intestine and enhance its lymphatic transport, thereby improving the oral bioavailability. Altogether, NNE is a promising carrier for the oral delivery of drugs with significant first-pass metabolism.Keywords: non-lipolysis nanoemulsion, raloxifene, first-pass metabolism, stability, bioavailability, endocytosis

Legionella pneumonia is a relatively rare but extremely progressive pulmonary infection with high mortality. Traditional cultural isolation remains the gold standard for the diagnosis of Legionella pneumonia. However, its harsh culture conditions, long turnaround time, and suboptimal sensitivity do not meet the clinical need for rapid and accurate diagnosis, especially for critically ill patients. So far, pathogenic detection techniques including serological assays, urinary antigen tests, and mass spectrometry, as well as nucleic acid amplification technique, have been developed, and each has its own advantages and limitations. This review summarizes the clinical characteristics and imaging findings of Legionella pneumonia, then discusses the advances, advantages, and limitations of the various pathogenetic detection techniques used for Legionella pneumonia diagnosis. The aim is to provide rapid and accurate guiding options for early identification and diagnosis of Legionella pneumonia in clinical practice, further easing healthcare burden.

Jingyi Ye,1,* Huiyi Wu,1,* Chuanli Huang,1 Wanting Lin,2 Caifeng Zhang,1 Bei Huang,2 Banyi Lu,2 Hongyu Xu,2 Xiaoling Li,3 Xiaoying Long1,41Department of Pharmaceutics, School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, People’s Republic of China; 2Department of Pharmacy of Chinese Materia Medica, School of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou 510006, People’s Republic of China; 3Department of Pharmaceutics and Medicinal Chemistry, Thomas J. Long School of Pharmacy & Health Sciences, University of the Pacific, Stockton, CA 95211, USA; 4Department of Oral Delivery, Guangdong Engineering and Technology Research Center of Topical Precise Drug Delivery System, Guangdong Pharmaceutical University, Guangzhou 510006, People’s Republic of China*These authors contributed equally to this workPurpose: The objective of this study was to compare the in vitro Fick’s first law, in vitro lipolysis, and in vivo rat assays for oral absorption of Biopharmaceutical Classification Systems Class II (BCS II) drugs in self-nanoemulsifying drug delivery system (SNEDDS), and studied drugs and oils properties effects on the absorption.Methods: The transport abilities of griseofulvin (GRI), phenytoin (PHE), indomethacin (IND), and ketoprofen (KET) in saturated water solutions and SNEDDS were investigated using the in vitro Madin-Darby canine kidney cell model. GRI and cinnarizine (CIN) in medium-chain triglycerides (MCT)-SNEDDS and long-chain triglycerides (LCT)-SNEDDS were administered in the in vivo SD rat and in vitro lipolysis models to compare the oral absorption and the distribution behaviors in GIT and build an in vitro-in vivo correlation (IVIVC).Results: In the cell model, the solubility of GRI, PHE, IND, and KET increased 6–8 fold by SNEDDS, but their permeability were only 18%, 4%, 8%, and 33% of those of their saturated water solutions, respectively. However, in vivo absorption of GRI-SNEDDS was twice that of the GRI suspension and those of CIN-SNEDDS were 15–21 fold those of the CIN suspension. In the lipolysis model, the GRI% in aqueous and pellet phases of MCT were similar to that in LCT. In contrast, the CIN% in the aqueous and pellet phases were decreased but that of the lipid phase increased. In addition, an IVIVC was found between the CIN% in the lipid phase and in vivo relative oral bioavailability (Fr).Conclusion: The in vitro cell model was still a suitable tool to study drug properties effects on biofilm transport and SNEDDS absorption mechanisms. The in vitro lipolysis model provided superior oral absorption simulation of SNEDDS and helped to build correlation with in vivo rats. The oral drug absorption was affected by drug and oil properties in SNEDDS.Keywords: SNEDDS, MDCK cell model, in vivo rat model, in vitro lipolysis, in vitro-in vivo correlation

Natalie Duong,1 Kevin Curley,1 Annie Brown,1 Alexander Campanelli,1 Mai Anh Do,1 Daniel Levy,1 Adarsh Tantry,1 Gerard Marriott,2 Biao Lu11Department of Bioengineering, School of Engineering, Santa Clara University, Santa Clara, CA 95053, USA; 2Department of Bioengineering and Tsinghua-Berkeley Shenzhen Institute, University of California at Berkeley, Berkeley, CA 94720, USABackground: Exosomes are ubiquitous naturally secreted stable nanovesicles that can be engineered to target and deliver novel therapeutics to treat a host of human diseases.Methods: We engineered the surfaces of cell-derived nanovesicles to act as decoys in the treatment of inflammation by antagonizing the major proinflammatory cytokine, tumor necrosis factor alpha (TNFα).Results: Decoy exosomes were generated by displaying the TNFα binding domain of human TNF receptor-1 (hTNFR1) on the outer surface of exosomes using stably transfected HEK293 cells. We developed an efficient method to purify the engineered exosomes from conditioned medium based on sequential centrifugation, ultrafiltration, and precipitation. We characterized decoy exosomes using immune-quantification, nanoparticle tracking analysis, and confocal microscopy to confirm that they retain the correct orientation, size, and shape of naturally produced exosomes. We demonstrated the engineered decoy exosomes specifically antagonize activities of TNFα using an inflammatory reporter cell line.Conclusions: Decoy exosomes produced in human cells serve as a novel biologic reagent for antagonizing inflammatory signaling mediated by TNFα.Keywords: Decoy exosomes, Inflammation, CD63, Receptor, TNFα, Drug delivery

Dao-Yu Zhu,1,* Bin Lu,2,* Jun-Hui Yin,1 Qin-Fei Ke,2 He Xu,2 Chang-Qing Zhang,1 Ya-Ping Guo,2 You-Shui Gao1,3 1Department of Orthopedic Surgery, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, China; 2The Education Ministry Key Lab of Resource Chemistry, Shanghai Key Laboratory of Rare Earth Functional Materials, Shanghai Normal University, Shanghai 200234, China; 3Centre for Orthopaedic Research, Faculty of Health and Medical Sciences, The University of Western Australia, Perth, WA 6009, Australia *These authors contributed equally to this work Background: Biomaterial-induced osteogenesis is mainly related to hierarchically porous structures and bioactive components. Rare earth elements are well known to promote osteogenesis and stimulate bone repair; however, the underlying biological effects of gadolinium (Gd) element on bone regeneration are not yet known. Methods: In this study, we successfully fabricated gadolinium-doped bioglass (Gd-BG) scaffolds by combining hollow mesoporous Gd-BG microspheres with chitosan and evaluated in vitro effects and underlying mechanisms with Cell Counting Kit-8, scanning electron microscopy, alkaline phosphatase, Alizarin red staining, and polymerase chain reaction. Cranial defect model of rats was constructed to evaluate their in vivo effects. Results: The results indicated that Gd-BG scaffolds could promote the proliferation and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs). Mechanistically, the Akt/GSK3β signaling pathway was activated by the Gd-BG scaffolds. The enhancing effect of Gd-BG scaffolds on the osteogenic differentiation of hBMSCs was inhibited by the addition of LY294002, an inhibitor of Akt. Moreover, the in vivo cranial defect model of rats indicated that the Gd-BG scaffolds could effectively promote bone regeneration. Conclusion: Both in vitro and in vivo results suggested that Gd-BG scaffolds have promising applications in bone tissue engineering. Keywords: bone scaffold, gadolinium, Akt/GSK3β pathway, bone regeneration

Background: Radioresistance is the major cause of cancer treatment failure. Additionally, splicing dysregulation plays critical roles in tumorigenesis. However, the involvement of alternative splicing in resistance of cancer cells to radiotherapy remains elusive. We sought to investigate the key role of the splicing factor SRSF1 in the radioresistance in lung cancer. Methods: Lung cancer cell lines, xenograft mice models, and RNA-seq were employed to study the detailed mechanisms of SRSF1 in lung cancer radioresistance. Clinical tumor tissues and TCGA dataset were utilized to determine the expression levels of distinct SRSF1-regulated splicing isoforms. KM-plotter was applied to analyze the survival of cancer patients with various levels of SRSF1-regulated splicing isoforms. Findings: Splicing factors were screened to identify their roles in radioresistance, and SRSF1 was found to be involved in radioresistance in cancer cells. The level of SRSF1 is elevated in irradiation treated lung cancer cells, whereas knockdown of SRSF1 sensitizes cancer cells to irradiation. Mechanistically, SRSF1 modulates various cancer-related splicing events, particularly the splicing of PTPMT1, a PTEN-like mitochondrial phosphatase. Reduced SRSF1 favors the production of short isoforms of PTPMT1 upon irradiation, which in turn promotes phosphorylation of AMPK, thereby inducing DNA double-strand break to sensitize cancer cells to irradiation. Additionally, the level of the short isoform of PTPMT1 is decreased in cancer samples, which is correlated to cancer patients' survival. Conclusions: Our study provides mechanistic analyses of aberrant splicing in radioresistance in lung cancer cells, and establishes SRSF1 as a potential therapeutic target for sensitization of patients to radiotherapy. Keywords: Alternative splicing, SRSF1, PTPMT1, AMPK, Radio-resistance

Objective To our knowledge, the independent association between central obesity, defined by waist circumference (WC) or waist-to-hip ratio (WHR), and diabetic retinopathy (DR) remains unknown in Chinese individuals. Method The study was conducted in two stages. First, the relationship between WC or WHR and DR was estimated in a case-control set (DR vs. non-DR) for the whole population before and after propensity score matching. Subsequently, a systematic review and meta-analysis was performed on evidence from the literature to validate the relationship. Results Of 511 eligible patients, DR (N = 156) and non-DR (N = 156) patients with similar propensity scores were included in the propensity score matching analyses. Central obesity (defined by WC) was associated with risk of DR (odds ratio [OR] 1.07, 95% confidence interval [95% CI] (1.03–1.10). The meta-analysis showed that central obesity significantly increased the risk of DR by 12% (OR 1.12, 95% CI 1.02–1.22). Analysis of data from 18 studies showed a significant association between continuous body mass index and risk of proliferative DR (OR 0.95, 95% CI 0.93–0.98; I 2 = 50%). Conclusion Central obesity, particularly as defined by WC, is associated with the risk of DR in the Chinese population.

Conary Meyer,1,* Joseph Losacco,1,* Zachary Stickney,1 Lingxuan Li,2 Gerard Marriott,3 Biao Lu1 1Department of Bioengineering, Santa Clara University, Santa Clara, 2Crown College, University of California at Santa Cruz, Santa Cruz, 3Department of Bioengineering, University of California at Berkeley, Berkeley, CA, USA *These authors contributed equally to this work Abstract: Exosomes are cell-derived nanovesicles that hold promise as living vehicles for intracellular delivery of therapeutics to mammalian cells. This potential, however, is undermined by the lack of effective methods to load exosomes with therapeutic proteins and to facilitate their uptake by target cells. Here, we demonstrate how a vesicular stomatitis virus glycoprotein (VSVG) can both load protein cargo onto exosomes and increase their delivery ability via a pseudotyping mechanism. By fusing a set of fluorescent and luminescent reporters with VSVG, we show the successful targeting and incorporation of VSVG fusions into exosomes by gene transfection and fluorescence tracking. We subsequently validate our system by live cell imaging of VSVG and its participation in endosomes/exosomes that are ultimately released from transfected HEK293 cells. We show that VSVG pseudotyping of exosomes does not affect the size or distributions of the exosomes, and both the full-length VSVG and the VSVG without the ectodomain are shown to integrate into the exosomal membrane, suggesting that the ectodomain is not required for protein loading. Finally, exosomes pseudotyped with full-length VSVG are internalized by multiple-recipient cell types to a greater degree compared to exosomes loaded with VSVG without the ectodomain, confirming a role of the ectodomain in cell tropism. In summary, our work introduces a new genetically encoded pseudotyping platform to load and enhance the intracellular delivery of therapeutic proteins via exosome-based vehicles to target cells. Keywords: exosome, VSVG, protein delivery, nanotechnology

Sen Jia,1,* Xinjie Yang,1,* Wen Song,2,* Lei Wang,1 Kaixiu Fang,3 Zhiqiang Hu,1,4 Zihui Yang,1 Chun Shan,1 Delin Lei,1 Bin Lu1 1Department of Oral and Maxillofacial Surgery, 2Department of Prosthetic Dentistry, 3Department of Implant Dentistry, School of Stomatology, State Key Laboratory of Military Stomatology, Fourth Military Medical University, Xi’an People’s Republic of China; 4Department of Otorhinolaryngology, No 113 Hospital of People’s Liberation Army, Ningbo, People’s Republic of China *These authors contributed to this paper equally and are considered to be joint first authors Abstract: Engineered bone substitutes are being extensively explored in response to growing demand. However, the angiogenesis that occurs during bone formation is often overlooked in scaffold design. In this novel study, we incorporated two small interfering RNAs (siRNAs), ie, small interfering RNA targets casein kinase 2 interaction protein 1 (siCkip-1) and small interfering RNA targets soluble VEGF receptor 1 (siFlt-1), which can promote osteogenesis and angiogenesis, into a chitosan sponge. This scaffold could maintain siRNAs for over 2 weeks in neutral phosphate-buffered saline and degraded rapidly in the presence of lysozyme. The chitosan sponge with siCkip-1 and siFlt-1 in vitro bioactivity was investigated using mesenchymal stem cells. Target genes were significantly suppressed, and osteocalcin, alkaline phosphatase, and vascular endothelial growth factor were significantly upregulated. Alizarin Red staining revealed that mineralization of the extracellular matrix was markedly enhanced by dual transfection. Further analysis by immunofluorescence confirmed that the siRNA-modified scaffold simultaneously improved the expression of osteocalcin and von Willebrand factor. In vivo testing in a skull critical-size defect model showed marked bone regeneration in rats treated with siCkip-1 and siFlt-1. In conclusion, chitosan sponge containing osteogenic and angiogenic siRNAs may be used as a scaffold for bone regeneration. The dual siRNA concept may also be useful in the biofunctionalization of other materials. Keywords: chitosan sponge, osteogenesis, angiogenesis, small interfering RNA

Elucidating the mechanism of over and under expression of proteins is critical in developing a better understanding of cancer. Multiple techniques are used to examine differential expression of proteins in cells and assess changes in protein expression in response to therapies such as radiation. Reduced expression can be caused by protein inactivation, mRNA instability, or reduced transcription. The following protocol was used to determine the mechanism for the reduced expression of an antiapoptotic factor, survivin, in normal tissues in response to radiation and the defect in cancer cells that prevents this reduction. We also examined ways to overcome survivin over expression in cancer cells in order to sensitize them to radiation. We will focus on the use of antisense oligonucleotides, cell cycle analysis, and luciferase reporter genes.

Abstract Background TALEs (transcription activator-like effectors) are powerful molecules that have broad applications in genetic and epigenetic manipulations. The simple design of TALEs, coupled with high binding predictability and specificity, is bringing genome engineering power to the standard molecular laboratory. Currently, however, custom TALE assembly is either costly or limited to few research centers, due to complicated assembly protocols, long set-up time and specific training requirements. Results We streamlined a Golden Gate-based method for custom TALE assembly. First, by providing ready-made, quality-controlled monomers, we eliminated the procedures for error-prone and time-consuming set-up. Second, we optimized the protocol toward a fast, two-day assembly of custom TALEs, based on four thermocycling reactions. Third, we increased the versatility for diverse downstream applications by providing series of vector sets to generate both TALENs (TALE nucleases) and TALE-TFs (TALE-transcription factors) under the control of different promoters. Finally, we validated our system by assembling a number of TALENs and TALE-TFs with DNA sequencing confirmation. We further demonstrated that an assembled TALE-TF was able to transactivate a luciferase reporter gene and a TALEN pair was able to cut its target. Conclusions We established and validated a do-it-yourself system that enables individual researchers to assemble TALENs and TALE-TFs within 2 days. The simplified TALE assembly combined with multiple choices of vectors will facilitate the broad use of TALE technology.

Abstract Background Transcription activator-like effectors (TALEs) are a class of naturally occurring transcription effectors that recognize specific DNA sequences and modulate gene expression. The modularity of TALEs DNA binding domain enables sequence-specific perturbation and offers broad applications in genetic and epigenetic studies. Although the efficient construction of TALEs has been established, robust functional tools to assess their functions remain lacking. Results We established a dual reporter system that was specifically designed for real-time monitoring and quantifying gene expression mediated by TALEs. We validated both sensitivity and specificity of this dual-reporter system in mammalian cells, and demonstrated that this dual reporter system is robust and potentially amenable to high throughput (HTP) applications. Conclusion We have designed, constructed and validated a novel dual reporter system for assessing TALE mediated gene regulations. This system offers a robust and easy-to- use tool for real-time monitoring and quantifying gene expression in mammalian cells.