Army Medical University

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is thatthere is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the completeprocess including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increasedautophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in manycases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as forreviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multipleassays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagyrelated protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

FASTA and FASTQ are basic and ubiquitous formats for storing nucleotide and protein sequences. Common manipulations of FASTA/Q file include converting, searching, filtering, deduplication, splitting, shuffling, and sampling. Existing tools only implement some of these manipulations, and not particularly efficiently, and some are only available for certain operating systems. Furthermore, the complicated installation process of required packages and running environments can render these programs less user friendly. This paper describes a cross-platform ultrafast comprehensive toolkit for FASTA/Q processing. SeqKit provides executable binary files for all major operating systems, including Windows, Linux, and Mac OSX, and can be directly used without any dependencies or pre-configurations. SeqKit demonstrates competitive performance in execution time and memory usage compared to similar tools. The efficiency and usability of SeqKit enable researchers to rapidly accomplish common FASTA/Q file manipulations. SeqKit is open source and available on Github at https://github.com/shenwei356/seqkit.

BACKGROUND: Osimertinib is an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) that is selective for both EGFR-TKI sensitizing and T790M resistance mutations in patients with non-small-cell lung cancer. The efficacy of osimertinib as compared with platinum-based therapy plus pemetrexed in such patients is unknown. METHODS: In this randomized, international, open-label, phase 3 trial, we assigned 419 patients with T790M-positive advanced non-small-cell lung cancer, who had disease progression after first-line EGFR-TKI therapy, in a 2:1 ratio to receive either oral osimertinib (at a dose of 80 mg once daily) or intravenous pemetrexed (500 mg per square meter of body-surface area) plus either carboplatin (target area under the curve, 5 [AUC5]) or cisplatin (75 mg per square meter) every 3 weeks for up to six cycles; maintenance pemetrexed was allowed. In all the patients, disease had progressed during receipt of first-line EGFR-TKI therapy. The primary end point was investigator-assessed progression-free survival. RESULTS: The median duration of progression-free survival was significantly longer with osimertinib than with platinum therapy plus pemetrexed (10.1 months vs. 4.4 months; hazard ratio; 0.30; 95% confidence interval [CI], 0.23 to 0.41; P<0.001). The objective response rate was significantly better with osimertinib (71%; 95% CI, 65 to 76) than with platinum therapy plus pemetrexed (31%; 95% CI, 24 to 40) (odds ratio for objective response, 5.39; 95% CI, 3.47 to 8.48; P<0.001). Among 144 patients with metastases to the central nervous system (CNS), the median duration of progression-free survival was longer among patients receiving osimertinib than among those receiving platinum therapy plus pemetrexed (8.5 months vs. 4.2 months; hazard ratio, 0.32; 95% CI, 0.21 to 0.49). The proportion of patients with adverse events of grade 3 or higher was lower with osimertinib (23%) than with platinum therapy plus pemetrexed (47%). CONCLUSIONS: Osimertinib had significantly greater efficacy than platinum therapy plus pemetrexed in patients with T790M-positive advanced non-small-cell lung cancer (including those with CNS metastases) in whom disease had progressed during first-line EGFR-TKI therapy. (Funded by AstraZeneca; AURA3 ClinicalTrials.gov number, NCT02151981 .).

autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Triple-negative breast cancer (TNBC), a specific subtype of breast cancer that does not express estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor 2 (HER-2), has clinical features that include high invasiveness, high metastatic potential, proneness to relapse, and poor prognosis. Because TNBC tumors lack ER, PR, and HER2 expression, they are not sensitive to endocrine therapy or HER2 treatment, and standardized TNBC treatment regimens are still lacking. Therefore, development of new TNBC treatment strategies has become an urgent clinical need. By summarizing existing treatment regimens, therapeutic drugs, and their efficacy for different TNBC subtypes and reviewing some new preclinical studies and targeted treatment regimens for TNBC, this paper aims to provide new ideas for TNBC treatment.

BACKGROUND The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed great threat to human health. T cells play a critical role in antiviral immunity but their numbers and functional state in COVID-19 patients remain largely unclear. METHODS We retrospectively reviewed the counts of T cells and serum cytokine concentration from data of 522 patients with laboratory-confirmed COVID-19 and 40 healthy controls. In addition, the expression of T cell exhaustion markers were measured in 14 COVID-19 cases. RESULTS The number of total T cells, CD4+ and CD8+ T cells were dramatically reduced in COVID-19 patients, especially in patients requiring Intensive Care Unit (ICU) care. Counts of total T cells, CD8+T cells or CD4+T cells lower than 800/μL, 300/μL, or 400/μL, respectively, are negatively correlated with patient survival. T cell numbers are negatively correlated to serum IL-6, IL-10 and TNF-α concentration, with patients in decline period showing reduced IL-6, IL-10 and TNF-α concentrations and restored T cell counts. T cells from COVID-19 patients have significantly higher levels of the exhausted marker PD-1. Increasing PD-1 and Tim-3 expression on T cells was seen as patients progressed from prodromal to overtly symptomatic stages. CONCLUSIONS T cell counts are reduced significantly in COVID-19 patients, and the surviving T cells appear functionally exhausted. Non-ICU patients, with total T cells counts lower than 800/μL may still require aggressive intervention even in the immediate absence of more severe symptoms due to a high risk for further deterioration in condition.

The gut microbiota benefits humans via short-chain fatty acid (SCFA) production from carbohydrate fermentation, and deficiency in SCFA production is associated with type 2 diabetes mellitus (T2DM). We conducted a randomized clinical study of specifically designed isoenergetic diets, together with fecal shotgun metagenomics, to show that a select group of SCFA-producing strains was promoted by dietary fibers and that most other potential producers were either diminished or unchanged in patients with T2DM. When the fiber-promoted SCFA producers were present in greater diversity and abundance, participants had better improvement in hemoglobin A1c levels, partly via increased glucagon-like peptide-1 production. Promotion of these positive responders diminished producers of metabolically detrimental compounds such as indole and hydrogen sulfide. Targeted restoration of these SCFA producers may present a novel ecological approach for managing T2DM.

Abstract Objective To assess the prevalence of diabetes and its risk factors. Design Population based, cross sectional study. Setting 31 provinces in mainland China with nationally representative cross sectional data from 2015 to 2017. Participants 75 880 participants aged 18 and older—a nationally representative sample of the mainland Chinese population. Main outcome measures Prevalence of diabetes among adults living in China, and the prevalence by sex, regions, and ethnic groups, estimated by the 2018 American Diabetes Association (ADA) and the World Health Organization diagnostic criteria. Demographic characteristics, lifestyle, and history of disease were recorded by participants on a questionnaire. Anthropometric and clinical assessments were made of serum concentrations of fasting plasma glucose (one measurement), two hour plasma glucose, and glycated haemoglobin (HbA 1c ). Results The weighted prevalence of total diabetes (n=9772), self-reported diabetes (n=4464), newly diagnosed diabetes (n=5308), and prediabetes (n=27 230) diagnosed by the ADA criteria were 12.8% (95% confidence interval 12.0% to 13.6%), 6.0% (5.4% to 6.7%), 6.8% (6.1% to 7.4%), and 35.2% (33.5% to 37.0%), respectively, among adults living in China. The weighted prevalence of total diabetes was higher among adults aged 50 and older and among men. The prevalence of total diabetes in 31 provinces ranged from 6.2% in Guizhou to 19.9% in Inner Mongolia. Han ethnicity had the highest prevalence of diabetes (12.8%) and Hui ethnicity had the lowest (6.3%) among five investigated ethnicities. The weighted prevalence of total diabetes (n=8385) using the WHO criteria was 11.2% (95% confidence interval 10.5% to 11.9%). Conclusion The prevalence of diabetes has increased slightly from 2007 to 2017 among adults living in China. The findings indicate that diabetes is an important public health problem in China.

Dear Editor

This case report describes birth of an infant with elevated anti–SARS-CoV-2 IgM antibodies and cytokine levels to a mother with polymerase chain reaction–confirmed coronavirus disease 2019 (COVID-19) despite no physical contact.

Exosomes are extracellular vesicles secreted by most eukaryotic cells and participate in intercellular communication. The components of exosomes, including proteins, DNA, mRNA, microRNA, long noncoding RNA, circular RNA, etc., which play a crucial role in regulating tumor growth, metastasis, and angiogenesis in the process of cancer development, and can be used as a prognostic marker and/or grading basis for tumor patients. Hereby, we mainly summarized as followed: the role of exosome contents in cancer, focusing on proteins and noncoding RNA; the interaction between exosomes and tumor microenvironment; the mechanisms that epithelial-mesenchymal transition, invasion and migration of tumor affected by exosomes; and tumor suppression strategies based on exosomes. Finally, the application potential of exosomes in clinical tumor diagnosis and therapy is prospected, which providing theoretical supports for using exosomes to serve precise tumor treatment in the clinic.

T-cell exhaustion was originally identified during chronic infection in mice, and was subsequently observed in humans with cancer. The exhausted T cells in the tumor microenvironment show overexpressed inhibitory receptors, decreased effector cytokine production and cytolytic activity, leading to the failure of cancer elimination. Restoring exhausted T cells represents an inspiring strategy for cancer treatment, which has yielded promising results and become a significant breakthrough in the cancer immunotherapy. In this review, we overview the updated understanding on the exhausted T cells in cancer and their potential regulatory mechanisms and discuss current therapeutic interventions targeting exhausted T cells in clinical trials.

Reactive oxygen species (ROS) can attack a diverse range of targets to exert antimicrobial activity, which accounts for their versatility in mediating host defense against a broad range of pathogens. Most ROS are formed by the partial reduction in molecular oxygen. Four major ROS are recognized comprising superoxide (O2•-), hydrogen peroxide (H2O2), hydroxyl radical (•OH), and singlet oxygen ((1)O2), but they display very different kinetics and levels of activity. The effects of O2•- and H2O2 are less acute than those of •OH and (1)O2, because the former are much less reactive and can be detoxified by endogenous antioxidants (both enzymatic and nonenzymatic) that are induced by oxidative stress. In contrast, no enzyme can detoxify •OH or (1)O2, making them extremely toxic and acutely lethal. The present review will highlight the various methods of ROS formation and their mechanism of action. Antioxidant defenses against ROS in microbial cells and the use of ROS by antimicrobial host defense systems are covered. Antimicrobial approaches primarily utilizing ROS comprise both bactericidal antibiotics and nonpharmacological methods such as photodynamic therapy, titanium dioxide photocatalysis, cold plasma, and medicinal honey. A brief final section covers reactive nitrogen species and related therapeutics, such as acidified nitrite and nitric oxide-releasing nanoparticles.

Macrophages are widely distributed innate immune cells that play indispensable roles in the innate and adaptive immune response to pathogens and in-tissue homeostasis. Macrophages can be activated by a variety of stimuli and polarized to functionally different phenotypes. Two distinct subsets of macrophages have been proposed, including classically activated (M1) and alternatively activated (M2) macrophages. M1 macrophages express a series of proinflammatory cytokines, chemokines, and effector molecules, such as IL-12, IL-23, TNF-α, iNOS and MHCI/II. In contrast, M2 macrophages express a wide array of anti-inflammatory molecules, such as IL-10, TGF-β, and arginase1. In most tumors, the infiltrated macrophages are considered to be of the M2 phenotype, which provides an immunosuppressive microenvironment for tumor growth. Furthermore, tumor-associated macrophages secrete many cytokines, chemokines, and proteases, which promote tumor angiogenesis, growth, metastasis, and immunosuppression. Recently, it was also found that tumor-associated macrophages interact with cancer stem cells. This interaction leads to tumorigenesis, metastasis, and drug resistance. So mediating macrophage to resist tumors is considered to be potential therapy.

The lungs from novel coronavirus pneumonia patients manifest significant pathological lesions, including the alveolar exudative inflammation and interstitial inflammation, alveolar epithelium proliferation and hyaline membrane formation. While the 2019-nCoV is mainly distributed in lung, the infection also involves in the damages of heart, vessels, liver, kidney and other organs. Further studies are warranted to investigate the mechanism underlying pathological changes of this disease.

A thorough understanding of the antimicrobial mechanisms of graphene materials (GMs) is critical to the manipulation of highly efficient antimicrobial nanomaterials for future biomedical applications. Here we review the most recent studies of GM-mediated antimicrobial properties. This review covers the physicochemical properties of GMs, experimental surroundings, and selected microorganisms as well as the interaction between GMs and selected microorganisms to explore controversial antimicrobial activities. Finally, we rationally analyze the strengths and weaknesses of the proposed mechanisms and provide new insights into the remaining challenges and perspectives for future studies.

Growing evidences suggest that the fibroblast growth factor/FGF receptor (FGF/FGFR) signaling has crucial roles in a multitude of processes during embryonic development and adult homeostasis by regulating cellular lineage commitment, differentiation, proliferation, and apoptosis of various types of cells. In this review, we provide a comprehensive overview of the current understanding of FGF signaling and its roles in organ development, injury repair, and the pathophysiology of spectrum of diseases, which is a consequence of FGF signaling dysregulation, including cancers and chronic kidney disease (CKD). In this context, the agonists and antagonists for FGF-FGFRs might have therapeutic benefits in multiple systems.

Previous studies suggest that gut microbiota is associated with neuropsychiatric disorders, such as Parkinson's disease, amyotrophic lateral sclerosis, and depression. However, whether the composition and diversity of gut microbiota is altered in patients with Alzheimer's disease (AD) remains largely unknown. In the present study, we collected fecal samples from 43 AD patients and 43 age- and gender-matched cognitively normal controls. 16S ribosomal RNA sequencing technique was used to analyze the microbiota composition in feces. The composition of gut microbiota was different between the two groups. Several bacteria taxa in AD patients were different from those in controls at taxonomic levels, such as Bacteroides, Actinobacteria, Ruminococcus, Lachnospiraceae, and Selenomonadales. Our findings suggest that gut microbiota is altered in AD patients and may be involved in the pathogenesis of AD.

Fracture-related infection (FRI) is a common and serious complication in trauma surgery. Accurately estimating the impact of this complication has been hampered by the lack of a clear definition. The absence of a working definition of FRI renders existing studies difficult to evaluate or compare. In order to address this issue, an expert group comprised of a number of scientific and medical organizations has been convened, with the support of the AO Foundation, in order to develop a consensus definition. The process that led to this proposed definition started with a systematic literature review, which revealed that the majority of randomized controlled trials in fracture care do not use a standardized definition of FRI. In response to this conclusion, an international survey on the need for and key components of a definition of FRI was distributed amongst all registered AOTrauma users. Approximately 90% of the more than 2000 surgeons who responded suggested that a definition of FRI is required. As a final step, a consensus meeting was held with an expert panel. The outcome of this process led to a consensus definition of FRI. Two levels of certainty around diagnostic features were defined. Criteria could be confirmatory (infection definitely present) or suggestive. Four confirmatory criteria were defined: Fistula, sinus or wound breakdown; Purulent drainage from the wound or presence of pus during surgery; Phenotypically indistinguishable pathogens identified by culture from at least two separate deep tissue/implant specimens; Presence of microorganisms in deep tissue taken during an operative intervention, as confirmed by histopathological examination. Furthermore, a list of suggestive criteria was defined. These require further investigations in order to look for confirmatory criteria. In the current paper, an overview is provided of the proposed definition and a rationale for each component and decision. The intention of establishing this definition of FRI was to offer clinicians the opportunity to standardize clinical reports and improve the quality of published literature. It is important to note that the proposed definition was not designed to guide treatment of FRI and should be validated by prospective data collection in the future.

Oxidative stress is associated with many acute and chronic inflammatory diseases, yet limited treatment is currently available clinically. The development of enzyme-mimicking nanomaterials (nanozymes) with good reactive oxygen species (ROS) scavenging ability and biocompatibility is a promising way for the treatment of ROS-related inflammation. Herein we report a simple and efficient one-step development of ultrasmall Cu5.4O nanoparticles (Cu5.4O USNPs) with multiple enzyme-mimicking and broad-spectrum ROS scavenging ability for the treatment of ROS-related diseases. Cu5.4O USNPs simultaneously possessing catalase-, superoxide dismutase-, and glutathione peroxidase-mimicking enzyme properties exhibit cytoprotective effects against ROS-mediated damage at extremely low dosage and significantly improve treatment outcomes in acute kidney injury, acute liver injury and wound healing. Meanwhile, the ultrasmall size of Cu5.4O USNPs enables rapid renal clearance of the nanomaterial, guaranteeing the biocompatibility. The protective effect and good biocompatibility of Cu5.4O USNPs will facilitate clinical treatment of ROS-related diseases and enable the development of next-generation nanozymes. Oxidative stress is involved in several diseases and is a target for intervention. Here, the authors report on the synthesis of ultrasmall copper-based nanozymes as reactive oxygen species scavengers and demonstrate improved treatment outcomes in acute liver and kidney injury and wound healing in vivo.