Idiopathic pulmonary fibrosis (IPF) is a fatal chronic interstitial lung disease with no established treatment and is characterized by progressive scarring of the lung tissue and an irreversible decline in lung function. Chronic inflammation has been demonstrated to be the pathological basis of fibrosis. Emerging studies have revealed that most interleukin-17 (IL-17) isoforms are essential for the mediation of acute and chronic inflammation via innate and adaptive immunity. Overexpression or aberrant expression of IL-17 cytokines contributes to various pathological outcomes, including the initiation and exacerbation of IPF. Here, we aim to provide an overview of IL-17 family members in the pathogenesis of IPF.

Bacteria can evolve rapidly by acquiring new traits such as virulence, metabolic properties, and most importantly, antimicrobial resistance, through horizontal gene transfer (HGT). Multidrug resistance in bacteria, especially in Gram-negative organisms, has become a global public health threat often through the spread of mobile genetic elements. Conjugation represents a major form of HGT and involves the transfer of DNA from a donor bacterium to a recipient by direct contact. Conjugative plasmids, a major vehicle for the dissemination of antimicrobial resistance, are selfish elements capable of mediating their own transmission through conjugation. To spread to and survive in a new bacterial host, conjugative plasmids have evolved mechanisms to circumvent both host defense systems and compete with co-resident plasmids. Such mechanisms have mostly been studied in model plasmids such as the F plasmid, rather than in conjugative plasmids that confer antimicrobial resistance (AMR) in important human pathogens. A better understanding of these mechanisms is crucial for predicting the flow of antimicrobial resistance-conferring conjugative plasmids among bacterial populations and guiding the rational design of strategies to halt the spread of antimicrobial resistance. Here, we review mechanisms employed by conjugative plasmids that promote their transmission and establishment in Gram-negative bacteria, by following the life cycle of conjugative plasmids.

Background: Early diagnosis and classification of infections increase the cure rate while decreasing complications, which is significant for severe infections, especially for war surgery. However, traditional methods rely on laborious operations and bulky devices. On the other hand, point-of-care (POC) methods suffer from limited robustness and accuracy. Therefore, it is of urgent demand to develop POC devices for rapid and accurate diagnosis of infections to fulfill on-site militarized requirements.

Methods: We developed a wave-shaped microfluidic chip (WMC) assisted multiplexed detection platform (WMC-MDP). WMC-MDP reduces detection time and improves repeatability through premixing of the samples and reaction of the reagents. We further combined the detection platform with the streptavidin–biotin (SA-B) amplified system to enhance the sensitivity while using chemiluminescence (CL) intensity as signal readout. We realized simultaneous detection of C-reactive protein (CRP), procalcitonin (PCT), and interleukin-6 (IL-6) on the detection platform and evaluated the sensitivity, linear range, selectivity, and repeatability. Finally, we finished detecting 15 samples from volunteers and compared the results with commercial ELISA kits.

Results: Detection of CRP, PCT, and IL-6 exhibited good linear relationships between CL intensities and concentrations in the range of 1.25–40 μg/ml, 0.4–12.8 ng/ml, and 50–1600 pg/ml, respectively. The limit of detection of CRP, PCT, and IL-6 were 0.54 μg/ml, 0.11 ng/ml, and 16.25 pg/ml, respectively. WMC-MDP is capable of good adequate selectivity and repeatability. The whole detection procedure takes only 22 min that meets the requirements of a POC device. Results of 15 samples from volunteers were consistent with the results detected by commercial ELISA kits.

Conclusions: WMC-MDP allows simultaneous, rapid, and sensitive detection of CRP, PCT, and IL-6 with satisfactory selectivity and repeatability, requiring minimal manipulation. However, WMC-MDP takes advantage of being a microfluidic device showing the coefficients of variation less than 10% enabling WMC-MDP to be a type of point-of-care testing (POCT). Therefore, WMC-MDP provides a promising alternative to POCT of multiple biomarkers. We believe the practical application of WMC-MDP in militarized fields will revolutionize infection diagnosis for soldiers.

Background: Stemness and chemoresistance contribute to cervical cancer recurrence and metastasis. In the current study, we determined the relevant players and role of N⁶-methyladenine (m⁶A) RNA methylation in cervical cancer progression.

Methods: The roles of m⁶A RNA methylation and centromere protein K (CENPK) in cervical cancer were analyzed using bioinformatics analysis. Methylated RNA immunoprecipitation was adopted to detect m⁶A modification of CENPK mRNA. Human cervical cancer clinical samples, cell lines, and xenografts were used for analyzing gene expression and function. Immunofluorescence staining and the tumorsphere formation, clonogenic, MTT, and EdU assays were performed to determine cell stemness, chemoresistance, migration, invasion, and proliferation in HeLa and SiHa cells, respectively. Western blotting analysis, co-immunoprecipitation, chromatin immunoprecipitation, and luciferase reporter, cycloheximide chase, and cell fractionation assays were performed to elucidate the underlying mechanism.

Results: Bioinformatics analysis of public cancer datasets revealed firm links between m⁶A modification patterns and cervical cancer prognosis, especially through ZC3H13-mediated m⁶A modification of CENPK mRNA. CENPK expression was elevated in cervical cancer, associated with cancer recurrence, and independently predicts poor patient prognosis [hazard ratio=1.413, 95% confidence interval=1.078–1.853, P=0.012]. Silencing of CENPK prolonged the overall survival time of cervical cancer-bearing mice and improved the response of cervical cancer tumors to chemotherapy in vivo (P<0.001). We also showed that CENPK was directly bound to SOX6 and disrupted the interactions of CENPK with β-catenin, which promoted β-catenin expression and nuclear translocation, facilitated p53 ubiquitination, and led to activation of Wnt/β-catenin signaling, but suppression of the p53 pathway. This dysregulation ultimately enhanced the tumorigenic pathways required for cell stemness, DNA damage repair pathways necessary for cisplatin/carboplatin resistance, epithelial-mesenchymal transition involved in metastasis, and DNA replication that drove tumor cell proliferation.

Conclusions: CENPK was shown to have an oncogenic role in cervical cancer and can thus serve as a prognostic indicator and novel target for cervical cancer treatment.

Granulomatous lobular mastitis (GLM) is a rare and chronic benign inflammatory disease of the breast. Difficulties exist in the management of GLM for many front-line surgeons and medical specialists who care for patients with inflammatory disorders of the breast. This consensus is summarized to establish evidence-based recommendations for the management of GLM. Literature was reviewed using PubMed from January 1, 1971 to July 31, 2020. Sixty-six international experienced multidisciplinary experts from 11 countries or regions were invited to review the evidence. Levels of evidence were determined using the American College of Physicians grading system, and recommendations were discussed until consensus. Experts discussed and concluded 30 recommendations on historical definitions, etiology and predisposing factors, diagnosis criteria, treatment, clinical stages, relapse and recurrence of GLM. GLM was recommended as a widely accepted definition. In addition, this consensus introduced a new clinical stages and management algorithm for GLM to provide individual treatment strategies. In conclusion, diagnosis of GLM depends on a combination of history, clinical manifestations, imaging examinations, laboratory examinations and pathology. The approach to treatment of GLM should be applied according to the different clinical stage of GLM. This evidence-based consensus would be valuable to assist front-line surgeons and medical specialists in the optimal management of GLM.

Background: Urinary tract infections (UTI), urolithiasis, and benign prostatic hyperplasia (BPH) are three of the most common nonmalignant conditions in urology. However, there is still a lack of comprehensive and updated epidemiological data. This study aimed to investigate the disease burden of UTI, urolithiasis, and BPH in 203 countries and territories from 1990 to 2019.

Methods: Data were extracted from the Global Burden of Disease 2019, including incident cases, deaths, disability-adjusted life-years (DALYs) and corresponding age-standardized rate (ASR) from 1990 to 2019. Estimated annual percentage changes (EAPC) were calculated to evaluate the trends of ASR. The associations between disease burden and social development degrees were analyzed using a sociodemographic index (SDI).

Results: Compared with 1990, the incident cases of UTI, urolithiasis, and BPH increased by 60.40%, 48.57%, and 105.70% in 2019, respectively. The age-standardized incidence rate (ASIR) of UTI increased (EAPC=0.08), while urolithiasis (EAPC=–0.83) and BPH (EAPC=–0.03) decreased from 1990 to 2019. In 2019, the age-standardized mortality rate (ASMR) of UTI and urolithiasis were 3.13/100,000 and 0.17/100,000, respectively. BPH had the largest increase (110.56%) in DALYs in the past three decades, followed by UTI (68.89%) and urolithiasis (16.95%). The burden of UTI was mainly concentrated in South Asia and Tropical Latin America, while the burden of urolithiasis and BPH was recorded in Asia and Eastern Europe. Moreover, the ASIR and SDI of urolithiasis in high-SDI regions from 1990 to 2019 were negatively correlated, while the opposite trend was seen in low-SDI regions. In 2019, the ASIR of UTI in females was 3.59 times that of males, while the ASIR of urolithiasis in males was 1.96 times higher than that in females. The incidence was highest in the 30–34, 55–59, and 65–69 age groups among the UTI, urolithiasis, and BPH groups, respectively.

Conclusions: Over the past three decades, the disease burden has increased for UTI but decreased for urolithiasis and BPH. The allocation of medical resources should be based more on the epidemiological characteristics and geographical distribution of diseases.

Background: Triple-negative breast cancer (TNBC) is the most aggressive subtype and occurs in approximately 15%–20% of diagnosed breast cancers. TNBC is characterized by its highly metastatic and recurrent features, as well as a lack of specific targets and targeted therapeutics. Epidermal growth factor receptor (EGFR) is highly expressed in a variety of tumors, especially in TNBC. LR004-VC-MMAE is a new EGFR-targeting antibody–drug conjugate produced by our laboratory. This study aimed to evaluate its antitumor activities against EGFR-positive TNBC and further studied its possible mechanism of antitumor action.

Methods: LR004-VC-MMAE was prepared by coupling a cytotoxic payload (MMAE) to an anti-EGFR antibody (LR004) via a linker, and the drug-to-antibody ratio (DAR) was analyzed by HIC-HPLC. The gene expression of EGFR in a series of breast cancer cell lines was assessed using a publicly available microarray dataset (GSE41313) and Western blotting. MDA-MB-468 and MDA-MB-231 cells were treated with LR004-VC-MMAE (0, 0.0066, 0.066, 0.66, 6.6 nmol/L), and the inhibitory effects of LR004-VC-MMAE on cell proliferation were examined by CCK-8 and colony formation. The migration and invasion capacity of MDA-MB-468 and MDA-MB-231 cells were tested at different LR004-VC-MMAE concentrations (2.5 and 5 nmol/L) with wound healing and Transwell invasion assays. Flow cytometric analysis and tumorsphere-forming assays were used to detect the killing effects of LR004-VC-MMAE on cancer stem cells (MDA-MB-468 and MDA-MB-231 cells). The mouse xenograft models were also used to evaluate the antitumor efficacy of LR004-VC-MMAE in vivo. Briefly, BALB/c nude mice were subcutaneously inoculated with MDA-MB-468 or MDAMB-231 cells. Then they were randomly divided into 4 groups (n=6 per group) and treated with PBS, naked LR004 (10 mg/kg), LR004-VC-MMAE (10 mg/kg), or doxorubicin, respectively. Tumor sizes and the body weights of mice were measured every 4 d. The effects of LR004-VC-MMAE on apoptosis and cell cycle distribution were analyzed by flow cytometry. Western blotting was used to detect the effects of LR004-VC-MMAE on EGFR, ERK, MEK phosphorylation and tumor stemness marker gene expression.

Results: LR004-VC-MMAE with a DAR of 4.02 were obtained. The expression of EGFR was found to be significantly higher in TNBC cells compared with non-TNBC cells (P<0.01). LR004-VC-MMAE inhibited the proliferation of EGFR-positive TNBC cells, and the IC₅₀ values of MDA-MB-468 and MDA-MB-231 cells treated with LR004-VC-MMAE for 72 h were (0.13±0.02) nmol/L and (0.66±0.06) nmol/L, respectively, which were significantly lower than that of cells treated with MMAE [(3.20±0.60) nmol/L, P<0.01, and (6.60±0.50) nmol/L, P<0.001]. LR004-VC-MMAE effectively inhibited migration and invasion of MDA-MB-468 and MDA-MB-231 cells. Moreover, LR004-VC-MMAE also killed tumor stem cells in EGFR-positive TNBC cells and impaired their tumorsphere-forming ability. In TNBC xenograft models, LR004-VC-MMAE at 10 mg/kg significantly suppressed tumor growth and achieved complete tumor regression on day 36. Surprisingly, tumor recurrence was not observed until the end of the experiment on day 52. In a mechanistic study, we found that LR004-VC-MMAE significantly induced cell apoptosis and cell cycle arrest at G₂/M phase in MDAMB-468 [(34±5)% vs. (12±2)%, P<0.001] and MDA-MB-231 [(27±4)% vs. (18±3)%, P<0.01] cells. LR004-VC-MMAE also inhibited the activation of EGFR signaling and the expression of cancer stemness marker genes such as Oct4, Sox2, KLF4 and EpCAM.

Conclusions: LR004-VC-MMAE showed effective antitumor activity by inhibiting the activation of EGFR signaling and the expression of cancer stemness marker genes. It might be a promising therapeutic candidate and provides a potential therapeutic avenue for the treatment of EGFR-positive TNBC.

Since the end of 2019, coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide. The RNA genome of SARS-CoV-2, which is highly infectious and prone to rapid mutation, encodes both structural and non-structural proteins. Vaccination is currently the only effective method to prevent COVID-19, and structural proteins are critical targets for vaccine development. Currently, many vaccines are in clinical trials or are already on the market. This review highlights ongoing advances in the design of prophylactic or therapeutic vaccines against COVID-19, including viral vector vaccines, DNA vaccines, RNA vaccines, live-attenuated vaccines, inactivated virus vaccines, recombinant protein vaccines and bionic nanoparticle vaccines. In addition to traditional inactivated virus vaccines, some novel vaccines based on viral vectors, nanoscience and synthetic biology also play important roles in combating COVID-19. However, many challenges persist in ongoing clinical trials.

Background: Disruption of the blood–brain barrier (BBB) after a stroke can lead to brain injury and neurological impairment. Previous work confirmed the involvement of the immunoproteasome subunit of low molecular mass peptide 2 (LMP2) in the pathophysiology of ischemia stroke. However, the relationship between the immunoproteasome LMP2 and the BBB remains unclear.

Methods: Adult male Sprague–Dawley rats were subjected to transient middle cerebral artery occlusion/reperfusion (MCAO/R). Three days before MCAO, the rats were treated with lentivirus-mediated LMP2 shRNA preparations by stereotactical injection into the ipsilateral hemispheric region. The rat brain microvascular endothelial cell (RBMVEC) line was exposed to oxygen–glucose deprivation/reperfusion (OGD/R) to mimic ischemic conditions in vitro. The RNA interference-mediated knockdown of LMP2 or _β-catenin was analysed in vivo and in vitro. Analysis of the quantity of extravasated Evans blue (EB) and cerebral fluorescent angiography were performed to evaluate the integrity of the BBB. Immunofluorescence and Western blotting were employed to detect the expression of target proteins. Cell migration was evaluated using a scratch migration assay. The results of immunofluorescence, Western blotting and cell migration were quantified using the software ImageJ (Version 1.53). Parametric data from different groups were compared using one-way ANOVA followed by the least significant difference (LSD) test.

Results: Cerebral ischemia led to lower levels of structural components of the BBB such as tight junction proteins [occludin, claudin-1 and zonula occludens (ZO-1)] in the MCAO/R group compared with the sham group (P<0.001). However, inhibition of the immunoproteasome LMP2 restored the expression of these proteins, resulting in higher levels of occludin, claudin-1 and ZO-1 in the LMP2-shRNA group compared with the control-shRNA group (P<0.001). In addition, inhibition of the immunoproteasome LMP2 contributed to higher microvascular density and decreased BBB permeability [e.g., the quantity of extravasated EB: LMP2-shRNA group (58.54±7.37) μg/g vs. control-shRNA group (103.74±4.32) μg/g, P<0.001], and promoted the upregulation of Wnt-3a and β-catenin proteins in rats following MCAO/R. In vitro experiments, OGD/R induced marked upregulation of LMP2, proapoptotic protein Bax and cleaved caspase-3, and downregulation of occludin, claudin-1, ZO-1 and Bcl-2, as well as inhibition of the Wnt/β-catenin pathway Wnt-3a and β-catenin proteins in RBMVECs, compared with the control group under normal culture conditions (P<0.001). However, silencing of LMP2 gene expression reversed these protein changes and promoted proliferation and migration of RBMVECs following OGD/R. Silencing of β-catenin by transfection of RBMVECs with β-catenin-siRNA aggravated the downregulation of tight junction proteins, and reduced the proliferation and migration of RBMVECs following OGD/R, compared with the control-siRNA group (P<0.001). LMP2-siRNA and β-catenin-siRNA co-transfection partly counteracted the beneficial effects of silencing LMP2-siRNA on the levels of tight junction proteins in RBMVECs exposed to OGD/R.

Conclusions: This study suggests that inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia induced BBB injury, and that the molecular mechanism involves the immunoproteasome-regulated activation of the Wnt/_β-catenin signalling pathway under ischemic conditions.

Background: Musculoskeletal injuries (MSkIs) are a leading cause of health care utilization, as well as limited duty and disability in the US military and other armed forces. MSkIs affect members of the military during initial training, operational training, and deployment and have a direct negative impact on overall troop readiness. Currently, a systematic overview of all risk factors for MSkIs in the military is not available.

Methods: A systematic literature search was carried out using the PubMed, Ovid/Medline, and Web of Science databases from January 1, 2000 to September 10, 2019. Additionally, a reference list scan was performed (using the "snowball method" ). Thereafter, an international, multidisciplinary expert panel scored the level of evidence per risk factor, and a classification of modifiable/non-modifiable was made.

Results: In total, 176 original papers and 3 meta-analyses were included in the review. A list of 57 reported potential risk factors was formed. For 21 risk factors, the level of evidence was considered moderate or strong. Based on this literature review and an in-depth analysis, the expert panel developed a model to display the most relevant risk factors identified, introducing the idea of the "order of importance" and including concepts that are modifiable/non-modifiable, as well as extrinsic/intrinsic risk factors.

Conclusions: This is the qualitative systematic review of studies on risk factors for MSkIs in the military that has attempted to be all-inclusive. A total of 57 different potential risk factors were identified, and a new, prioritizing injury model was developed. This model may help us to understand risk factors that can be addressed, and in which order they should be prioritized when planning intervention strategies within military groups.