New insights from bidirectional Mendelian randomization: causal relationships between telomere length and mitochondrial DNA copy number in aging biomarkers.

Mitochondrial DNA (mtDNA) copy number and telomere length (TL) are dynamic factors that have been linked to the aging process in organisms. However, the causal relationship between these variables remains uncertain. In this research, instrumental variables (IVs) related to mtDNA copy number and TL were obtained from publicly available genome-wide association studies (GWAS). Through bidirectional Mendelian randomization (MR) analysis, we examined the potential causal relationship between these factors. The forward analysis, with mtDNA copy number as the exposure and TL as the outcome, did not reveal a significant effect (B=-0.004, P>0.05). On the contrary, upon conducting a reverse analysis, it was found that there exists a positive causal relationship (B=0.054, P<0.05). Sensitivity analyses further confirmed the reliability of these results. The outcomes of this study indicate a one-way positive causal relationship, indicating that telomere shortening in the aging process may lead to a decrease in mtDNA copy number, providing new perspectives on their biological mechanisms.

Neutrophil extracellular traps (NETs) and Th-2 dominant immune responses in chronic granulomatous chromobalstomycosis.

Chromoblastomycosis (CBM), a chronic, granulomatous, suppurative mycosis of the skin and subcutaneous tissue, is caused by several dematiaceous fungi. The formation of granulomas, tissue proliferation, and fibrosis in response to these pathogenic fungi is believed to be intricately linked to host immunity. To understand this complex interaction, we conducted a comprehensive analysis of immune cell infiltrates, neutrophil extracellular traps (NETs) formation, and the fibrosis mechanism in 20 CBM lesion biopsies using immunohistochemical and immunofluorescence staining methods. The results revealed a significant infiltration of mixed inflammatory cells in CBM granulomas, prominently featuring a substantial presence of Th2 cells and M2 macrophages. These cells appeared to contribute to the production of collagen I and III in the late fibrosis mechanism, as well as NETs formation. The abundance of Th2 cytokines may act as a factor promoting the bias of macrophage differentiation toward M2, which hinders efficient fungal clearance while accelerates the proliferation of fibrous tissue. Furthermore, the expression of IL-17 was noted to recruit neutrophils, facilitating subsequent NETs formation within CBM granulomas to impede the spread of sclerotic cells. Understanding of these immune mechanisms holds promise for identifying therapeutic targets for managing chronic granulomatous CBM.

Case report: Clinical complete response in advanced ALK-positive lung squamous cell carcinoma: a case study of successful anti-PD-1 immunotherapy post ALK-TKIs failure.

In patients with advanced lung adenocarcinoma (LADC) harboring the echinoderm microtubule-associated protein-like 4 (EML4) -anaplastic lymphoma kinase (ALK) rearrangement, targeted therapy typically demonstrates superior efficacy as an initial treatment compared to chemotherapy. Following resistance to ALK-tyrosine kinase inhibitors (TKIs), regimens incorporating platinum-based dual agents or combined with bevacizumab often show effectiveness. However, therapeutic alternatives become constrained after resistance develops to both TKIs and platinum-based therapies. Given that the majority of ALK-positive non-small cell lung carcinomas (NSCLC) are LADC, the benefits of TKIs for patients with ALK-positive lung squamous cell carcinoma (LSCC) and the optimal treatment strategy for these patients remain a subject of debate. In this case study, we report on a patient with advanced LSCC, in whom the EML4-ALK rearrangement was identified via ARMS-PCR (Amplification Refractory Mutation System-Polymerase Chain Reaction). The patient underwent oral treatment with crizotinib and alectinib, showing effectiveness in both first-line and second-line ALK-TKI therapies, albeit with limited progression-free survival (PFS). Subsequent resistance to second-generation TKI was followed by the detection of tumors in the left neck region via computed tomography (CT). Biopsy pathology revealed non-squamous cell carcinoma, and subsequent treatment with platinum-based double-drug therapy proved ineffective. Further analysis through next-generation sequencing (NGS) indicated ALK negativity but a high expression of programmed death-ligand 1 (PD-L1). Immunotherapy was then initiated, resulting in a PFS of over 29 months and clinical complete remission (cCR). This case underscores the potential benefit of ALK-TKIs in patients with ALK-positive LSCC. Resistance to second-generation TKIs may lead to ALK negativity and histological transformation, highlighting the necessity of repeated biopsies post-TKI resistance for informed treatment decision-making. As of November 2023, imaging studies continue to indicate cCR in the patient, with a survival time exceeding 47 months.

A dynamic nomogram predicting symptomatic pneumonia in patients with lung cancer receiving thoracic radiation.

PURPOSE: The most common and potentially fatal side effect of thoracic radiation therapy is radiation pneumonitis (RP). Due to the lack of effective treatments, predicting radiation pneumonitis is crucial. This study aimed to develop a dynamic nomogram to accurately predict symptomatic pneumonitis (RP ≥ 2) following thoracic radiotherapy for lung cancer patients. METHODS: Data from patients with pathologically diagnosed lung cancer at the Zhongshan People's Hospital Department of Radiotherapy for Thoracic Cancer between January 2017 and June 2022 were retrospectively analyzed. Risk factors for radiation pneumonitis were identified through multivariate logistic regression analysis and utilized to construct a dynamic nomogram. The predictive performance of the nomogram was validated using a bootstrapped concordance index and calibration plots. RESULTS: Age, smoking index, chemotherapy, and whole lung V5/MLD were identified as significant factors contributing to the accurate prediction of symptomatic pneumonitis. A dynamic nomogram for symptomatic pneumonitis was developed using these risk factors. The area under the curve was 0.89(95% confidence interval 0.83-0.95). The nomogram demonstrated a concordance index of 0.89(95% confidence interval 0.82-0.95) and was well calibrated. Furthermore, the threshold values for high- risk and low- risk were determined to be 154 using the receiver operating curve. CONCLUSIONS: The developed dynamic nomogram offers an accurate and convenient tool for clinical application in predicting the risk of symptomatic pneumonitis in patients with lung cancer undergoing thoracic radiation.

Mycobacterium tuberculosis PE_PGRS45 (Rv2615c) Promotes Recombinant Mycobacteria Intracellular Survival via Regulation of Innate Immunity, and Inhibition of Cell Apoptosis.

Tuberculosis (TB), a bacterial infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis), is a significant global public health problem. Mycobacterium tuberculosis expresses a unique family of PE_PGRS proteins that have been implicated in pathogenesis. Despite numerous studies, the functions of most PE_PGRS proteins in the pathogenesis of mycobacterium infections remain unclear. PE_PGRS45 (Rv2615c) is only found in pathogenic mycobacteria. In this study, we successfully constructed a recombinant Mycobacterium smegmatis (M. smegmatis) strain which heterologously expresses the PE_PGRS45 protein. We found that overexpression of this cell wall-associated protein enhanced bacterial viability under stress in vitro and cell survival in macrophages. MS_PE_PGRS45 decreased the secretion of pro-inflammatory cytokines such as IL-1ß, IL-6, IL-12p40, and TNF-α. We also found that MS_PE_PGRS45 increased the expression of the anti-inflammatory cytokine IL-10 and altered macrophage-mediated immune responses. Furthermore, PE_PGRS45 enhanced the survival rate of M. smegmatis in macrophages by inhibiting cell apoptosis. Collectively, our findings show that PE_PGRS45 is a virulent factor actively involved in the interaction with the host macrophage.

Efficacy and Postoperative Complication Rates of Different Surgical Approaches for Calcaneal Fractures: A Network Meta-analysis.

Context: Calcaneal fractures (CFs) are the most common kind of tarsal fracture. The choice of surgical approach is a key element in the management of CFs, but the best method remains in dispute. Also, no single approach is appropriate for all kinds of CFs. Objective: The study intended to evaluate the relationship between six surgical approaches for clinical treatment of CFs and prevention of postoperative complications, to provide an evidence-based approach for treatment. Design: The research team performed a meta-analysis using the data from a previously published review and updating that data through a new narrative review. The team performed a systematic search in PubMed, Embase, the Cochrane Library, and the Chinese National Knowledge Internet (CNKI) from inception until January 2022, with no language restrictions. The search used the following keywords for the search: calcaneus, heel bone, surgical wounds, surgical incisions, prospective trials, prospective trials, and randomized controlled trials. Outcome Measures: The research team compared the complication rates, American Orthopedic Foot and Ankle Society (AOFAS) scores, and Bohler's angles for the six surgical approaches, which were: (1) the extensive lateral approach (ELA), (2) the sinus tarsi approach (STA), (3) the horizontal arc approach (HAA), (4) the longitudinal approach (LA), (5) the oblique lateral incision (OLI), and (6) the modified incision (MI)). The team summarized the results using a random effects model. Results: The research team analyzed the data from 19 RCTs with 1521 participants. They all were randomized controlled trials (RCTs). The complication rates were available for 18 studies, which included 1474 participants. The rates were significantly lower: (1) for HAA compared to ELA, [OR=-2.03; 95% CrI: [-3.63, -0.43)]; (2) for LA compared to ELA (OR=-1.83; 95% CrI: [-2.83, -0.84]); and (3) for STA compared to ELA (OR=- 1.22; 95% CrI: [-1.67, -0.78]). Of the 19 studies, 11 RCTs, with 942 participants, used the AOFAS scale. The probabilities for the surface under the cumulative ranking curve (SUCRA) indicated that OLI (0.694 ) >LA (0.596) >HAA (0.51) >STA (0.477) >ELA (0.224). In addition, ELA had the worst SUCRA (0.224). Of the 19 studies, 15 RCTs, with 1376 participants, used the Bohler angle as an outcome measure. The probability of SUCRA for the surgical approaches indicated that LA (0.723) >ELA (0.667) >STA (0.468) >HAA (0.373) >MI (0.27). Conclusions: The meta-analysis provides an evidence-based approach to the clinical treatment of CFs for six surgical approaches. HAA had the best outcomes, and ELA had the worst.

A case of acute myocarditis induced by PD-1 inhibitor (sintilimab) in the treatment of large cell neuroendocrine carcinoma.

The combination of Sintilimab with pemetrexed/platinum has become the first-line treatment for non-squamous non-small-cell lung carcinoma (NSCLC). Here, we report a patient with metastatic large cell neuroendocrine carcinoma (LCNEC) treated with Sintilimab for five cycles who developed shortness of breath after activity. The level of creatine kinase (CK), creatine kinase-MB (CK-MB) and cardiac troponin T (cTnT) were significantly increased. The cardiac MR suggested that heart function was slightly decreased. Considering that the patient did not take any illicit drugs, without history of autoimmune disease, coronary heart disease, arrhythmia, or chronic heart failure, we diagnosed the patient with Sintilimab-induced myocarditis. The symptoms alleviated after rapid use of glucocorticoids. Myocarditis is a rare immune-related adverse events (irAEs), especially myocarditis induced by programmed cell death receptor-1 (PD-1) inhibitor in the treatment of LCNEC.

Mycobacterium tuberculosis PE8 (Rv1040c) Promotes the Intracellular Survival of Recombinant Mycobacterium by Regulating Host Inflammatory Cytokines and Inhibiting Cell Late Apoptosis.

Tuberculosis is an important chronic and often fatal infectious disease mainly caused by the bacterium Mycobacterium tuberculosis (Mtb). Mtb is one of the most successful pathogens that harbors several potential virulence factors not found in nonpathogenic mycobacteria. As the Mtb cell envelope is closely associated with its virulence and resistance, it is very important to understand the cell envelope for better treatment of causative pathogen. There is increasing evidence that Pro-Glu (PE) and Pro-Pro-Glu (PPE) proteins are the major effectors of virulence and persistence encoded in the Mtb H37Rv genome. However, the function of PE8 has not been explored to date. In this study, we heterologously expressed PE8 in nonpathogenic, fast-growing M. smegmatis to investigate the interaction between PE8 and the host to determine its possible biological functions. We found that recombinant M. smegmatis cells expressing PE8 were less susceptible to sodium dodecyl sulfate-induced surface stress compared with those expressing the empty vector, suggesting that PE8 may be involved in stress responses. In addition, macrophages infected with PE8-expressing M. smegmatis produced obviously lower levels of the proinflammatory factor IL-1ß, IL-6, and TNF-α and higher levels of the inhibitory factor IL-10. We further found that PE8 promoted M. smegmatis survival within macrophages by inhibiting late apoptosis of macrophages. Collectively, selective targeting of the PE/PPE protein family offers an untapped opportunity to the development of more effective and safer drugs against Mtb infection.

Expression,purification and bioinformatics analysis of PE＿PGRS35 protein of Mycobacterium tuberculosis H37Rv strain / 中国生物制品学杂志

@#Abstract： Objective To clone PE_PGRS35 gene of Mycobacterium tuberculosis（MTB），construct recombinant vector pET28a⁃PE_PGRS35，express and purify the PE_PGRS35 protein of MTB H37Rv heterologously，and explore a new target against MTB after bioinformatics analysis. Methods The PE_PGRS35 coding gene was amplified by PCR and used to construct the expression vector pET28a⁃PE_PGRS35 by recombinant cloning technology，which was transformed to E. coli BL21（DE3）after successful sequencing and induced by using IPTG. The obtained PE_PGRS35 protein was purified by Ni column affinity chromatography and analyzed by bioinformatics. Results The pET28a⁃PE_PGRS35 prokaryotic expression vector was constructed correctly as identified by sequencing. The PE_PGRS35 protein was mainly expressed in the form of inclusion bodies，with a relative molecular mass of about 53 000 and a purity of 90%. Bioinformatics analysis showed that PE_PGRS35 protein was an acid⁃labile protein，with main secondary structure of β⁃sheet and random coil，and no transme⁃ mbrane region，which was presumed to be an extramembrane protein with 39 phosphorylation sites and two conserved domains. Total 10 proteins，including Rv1769，PPE8，PPE64，PPE54，PPE24，PPE16，PPE35，PPE6，PPE28 and PE2， interacted with PE_PGRS35 protein. Conclusion PE_PGRS35 protein with high purity was successfully obtained，which provided a reference for the further development of new targets for drugs against MTB.

Non-pharmaceutical treatments to relieve pain or reduce opioid analgesic intake and improve quality of life after total hip replacement: a meta analysis.

To reduce pain after total hip replacement (THR), researchers are interested in drug-free interventions. However, there is still a lack of consensus on their prevention efficacy. We performed a meta-analysis to evaluate the use of nonpharmaceutical interventions for postoperative pain management after THR. We searched the Cochrane Library, MEDLINE, EMBASE, Web of Science, PEDRO, and ClinicalTrials.gov databases for articles published between and 1991 and 2020. The main outcome measures were postoperative pain, opioid consumption, and quality of life (QoL). In total, 1,942 patients were studied. We found moderate evidence indicating postoperative pain relief measured by the Western Ontario and McMaster Universities Arthritis Index Scale, with mean differences (MDs) of -0.28 (95% confidence interval [CI], -0.49 to -0.07; P=0.01; I2 =0%) within three months, -0.19 (95% CI, -0.40 to 0.02; P=0.07; I2 =0%) between 3-6 months, and -0.13 (95% CI, -0.35 to 0.08; P=0.21; I2 =0%) between 6-12 months. Additionally, we found that acupuncture therapy could reduce opioid analgesic consumption (MD, -0.98; 95% CI, -1.18 to -0.79; fentanyl [mg/h]; P<0.01; I2 =72.2%) and significantly improve pain relief with an MD of 0.90 (95% CI, 0.47 to 1.33; P<0.01; I2 =0%) measured using the visual analog scale. Electrotherapy slightly improved perceived pain with an MD of 0.22 (95% CI, -0.27 to 0.70; P=0.37; I2 =0%). Moreover, moderate evidence has shown that preoperative exercises improve QoL. This meta-analysis suggested that continuous passive motion did not improve pain or QoL. Postoperative exercise was associated with pain relief and improved QoL. Acupuncture therapy after THR has been shown to reduce opioid analgesic consumption.

[Establishment of a THP-1 macrophage model infected by recombinant Mycobacterium smegmatis expressing green fluorescent protein].

Objective To establish a THP-1 macrophage model infected by Mycobacterium smegmatis expressing green fluorescent protein (GFP), to quickly locate and visually detect Mycobacterium smegmatis, and to provide a tracer tool to identify the pathogenesis of tuberculosis and develop new tuberculosis vaccines. Methods The enhanced green fluorescent protein (EGFP) gene sequence was amplified by PCR using pEGFP-N1 plasmid as a template to obtain the coding gene of EGFP, and the amplified product was cloned into the vector pALACE to establish the recombinant plasmid pALACE-EGFP. Electroporation transformed the pALACE-EGFP into Mycobacterium smegmatis, and recombinant Mycobacterium smegmatis clones were screened by hygromycin resistance. After expanded culture, the smears were observed by fluorescence microscopy. The THP-1 macrophages were infected with recombinant Mycobacterium smegmatis, and the expression of EGFP was observed. Results The recombinant plasmid pALACE-EGFP was constructed appropriately. The recombinant Mycobacterium smegmatis was observed under fluorescence microscope. And it was confirmed that EGFP was expressed in recombinant Mycobacterium smegmatis, and THP-1 macrophages emitted green fluorescence after infection. Conclusion The successful establishment of recombinant Mycobacterium smegmatis expressing EGFP protein provides insights for investigating infection and pathogenesis of Mycobacterium tuberculosis.

Hypoxia-Related Signature Is a Prognostic Biomarker of Pancreatic Cancer.

Background: Hypoxia plays a significant role in the pathogenesis of pancreatic cancer, but the effect of hypoxia-related genes in pancreatic cancer remains to be elucidated. This study aimed to identify hypoxia-related genes related to pancreatic cancer and construct a prognostic signature. Methods: Pancreatic cancer datasets were retrieved from TCGA database. Cox regression analyses were used to identify hypoxia-related genes and construct a prognostic signature. Datasets from International Cancer Genome Consortium and GEO databases were used as validated cohorts. The CIBERSORT method was applied to estimate the fractions of immune cell types. DNA methylation and protein levels of the genes in pancreatic cancer were examined. Results: Three hypoxia-related genes (TES, LDHA, and ANXA2) were identified as associated with patient survival and selected to construct a prognostic signature. Patients were divided into high- and low-risk groups based on the signature. Those in the high-risk group showed worse survival than those in the low-risk group. The signature was shown to be involved in the HIF-1 signaling pathway. The time-dependent ROC analyses of three independent validated cohorts further revealed that this signature had a better prognostic value in the prediction of the survival of pancreatic cancer patients. Immune cells analysis for three datasets demonstrated that high-risk signature was significantly associated with macrophages and T cells. DNA methylation and protein levels of the three genes validated their aberrant expression in pancreatic cancer. Conclusions: Our research provided a novel and reliable prognostic signature that composes of three hypoxia-related genes to estimate the prognosis of pancreatic cancer.

Deletion C-terminal thioesterase abolishes melanin biosynthesis, affects metabolism and reduces the pathogenesis of Fonsecaea monophora.

Dematiaceous Fonsecaea monophora is one of the major pathogens of chromoblastomycosis. It has been well established that melanization is catalyzed by the type I polyketide synthase (PKS) in F. monophora. Multidomain protein Type I PKS is encoded by six genes, in which the last enzyme thioesterase (TE) catalyzes the cyclization and releases polyketide. Two PKS genes AYO21_03016 (pks1) and AYO21_10638 have been found in F. monophora and both PKS loci have the same gene arrangement but the TE domain in AYO21_10638 is truncated at 3'- end. TE may be the key enzyme to maintain the function of pks1. To test this hypothesis, we constructed a 3'-end 500 bp deletion mutant of AYO21_03016 (Δpks1-TE-C500) and its complemented strain. We profiled metabolome of this mutant and analyzed the consequences of impaired metabolism in this mutant by fungal growth in vitro and by pathogenesis in vivo. Compared with wild-type strain, we found that the mutant repressed pks1 expression and other 5 genes expression levels were reduced by more than 50%, perhaps leading to a corresponding melanin loss. The mutant also reduced sporulation and delayed germination, became vulnerable to various environmental stresses and was less resistance to macrophage or neutrophil killings in vitro, and less virulence in mice footpad model. Metabolomic analysis indicated that many metabolites were remarkably affected in Δpks1-TE-C500, in particular, an increased nicotinamide and antioxidant glutathione. In conclusion, we confirmed the crucial role of C-terminal TE in maintaining fully function of pks1 in F. monophora. Deletion of TE negatively impacts on the synthesis of melanin and metabolites that eventually affect growth and virulence of F. monophora. Any potential inhibitor of TE then could be a novel antifungal target for drug development.

A prediction model for xerostomia in locoregionally advanced nasopharyngeal carcinoma patients receiving radical radiotherapy.

BACKGROUND: This study was to evaluate the predictors of xerostomia and Grade 3 xerostomia in locoregionally advanced nasopharyngeal carcinoma (NPC) patients receiving radical radiotherapy and establish prediction models for xerostomia and Grade 3 xerostomia based on the predictors. METHODS: Totally, 365 patients with locoregionally advanced NPC who underwent radical radiotherapy were randomly divided into the training set (n = 255) and the testing set (n = 110) at a ratio of 7:3. All variables were included in the least absolute shrinkage and selection operator regression to screen out the potential predictors for xerostomia as well as the Grade 3 xerostomia in locoregionally advanced NPC patients receiving radical radiotherapy. The random forest (RF), a decision tree classifier (DTC), and extreme-gradient boosting (XGB) models were constructed. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), area under the curve (AUC) and accuracy were analyzed to evaluate the predictive performance of the models. RESULTS: In the RF model for predicting xerostomia, the sensitivity was 1.000 (95%CI 1.000-1.000), the PPV was 0.990 (95%CI 0.975-1.000), the NPV was 1.000 (95%CI 1.000-1.000), the AUC was 0.999 (95%CI 0.997-1.000) and the accuracy was 0.992 (95%CI 0.981-1.000) in the training set. The sensitivity was 0.933 (95%CI 0.880-0.985), the PPV was 0.933 (95%CI 0.880-0.985), and the AUC was 0.915 (95%CI 0.860-0.970) in the testing set. Hypertension, age, total radiotherapy dose, dose at 50% of the left parotid volume, mean dose to right parotid gland, mean dose to oral cavity, and course of induction chemotherapy were important variables associated with the risk of xerostomia in locoregionally advanced NPC patients receiving radical radiotherapy. The AUC of DTC model for predicting xerostomia was 0.769 (95%CI 0.666-0.872) in the testing set. The AUC of the XGB model for predicting xerostomia was 0.834 (0.753-0.916) in the testing set. The RF model showed the good predictive ability with the AUC of 0.986 (95%CI 0.972-1.000) in the training set, and 0.766 (95%CI 0.626-0.905) in the testing set for identifying patients who at high risk of Grade 3 xerostomia in those with high risk of xerostomia. CONCLUSIONS: An RF model for predicting xerostomia in locoregionally advanced NPC patients receiving radical radiotherapy and an RF model for predicting Grade 3 xerostomia in those with high risk of xerostomia showed good predictive ability.

Exosomal HMGA2 protein from EBV-positive NPC cells destroys vascular endothelial barriers and induces endothelial-to-mesenchymal transition to promote metastasis.

Increased vascular permeability facilitates metastasis. Cancer-secreted exosomes are emerging mediators of cancer-host crosstalk. Epstein-Barr virus (EBV), identified as the first human tumor-associated virus, plays a crucial role in metastatic tumors, especially in nasopharyngeal carcinoma (NPC). To date, whether and how exosomes from EBV-infected NPC cells affect vascular permeability remains unclear. Here, we show that exosomes from EBV-positive NPC cells, but not exosomes from EBV-negative NPC cells, destroy endothelial cell tight junction (TJ) proteins, which are natural barriers against metastasis, and promote endothelial-to-mesenchymal transition (EndMT) in endothelial cells. Proteomic analysis revealed that the level of HMGA2 protein was higher in exosomes derived from EBV-positive NPC cells compared with that in exosomes derived from EBV-negative NPC cells. Depletion of HMGA2 in exosomes derived from EBV-positive NPC cells attenuates endothelial cell dysfunction and tumor cell metastasis. In contrast, exosomes from HMGA2 overexpressing EBV-negative NPC cells promoted these processes. Furthermore, we showed that HMGA2 upregulates the expression of Snail, which contributes to TJ proteins reduction and EndMT in endothelial cells. Moreover, the level of HMGA2 in circulating exosomes is significantly higher in NPC patients with metastasis than in those without metastasis and healthy negative controls, and the level of HMGA2 in tumor cells is associated with TJ and EndMT protein expression in endothelial cells. Collectively, our findings suggest exosomal HMGA2 from EBV-positive NPC cells promotes tumor metastasis by targeting multiple endothelial TJ and promoting EndMT, which highlights secreted HMGA2 as a potential therapeutic target and a predictive marker for NPC metastasis.

Molecular Characteristics of Regional Chromoblastomycosis in Guangdong, China: Epidemiological, Clinical, Antifungal Susceptibility, and Serum Cytokine Profiles of 45 Cases.

Chromoblastomycosis (CBM) is a chronic disease caused by several species of dematiaceous fungi. In this study, a regional collection of 45 CBM cases was conducted in Guangdong, China, a hyper-endemic area of CBM. Epidemiology findings indicated that the mean age of cases was 61.38 ± 11.20 years, long duration ranged from 3 months to 30 years, and the gender ratio of male to female was 4.6:1. Thirteen cases (29%) declared underlying diseases. Verrucous form was the most common clinical manifestation (n = 19, 42%). Forty-five corresponding clinical strains were isolated, and 28 of them (62%) were identified as F. monophora; the remaining 17 (38%) were identified as F. nubica through ITS rDNA sequence analysis. Antifungal susceptibility tests in vitro showed low MICs in azoles (PCZ 0.015-0.25 µg/ml, VCZ 0.015-0.5 µg/ml, and ITZ 0.03-0.5 µg/ml) and TRB (0.015-1 µg/ml). Itraconazole combined with terbinafine was the main therapeutic strategy used for 31 of 45 cases, and 68% (n = 21) of them improved or were cured. Cytokine profile assays indicated upregulation of IL-4, IL-7, IL-15, IL-11, and IL-17, while downregulation of IL-1RA, MIP-1ß, IL-8, and IL-16 compared to healthy donors (p < 0.05). The abnormal cytokine profiles indicated impaired immune response to eliminate fungus in CBM cases, which probably contributed to the chronic duration of this disease. In conclusion, we investigated the molecular epidemiological, clinical, and laboratory characteristics of CBM in Guangdong, China, which may assist further clinical therapy, as well as fundamental pathogenesis studies of CBM.

[Prokaryotic expression and identification of PPE15 protein from Mycobacterium tuberculosis and preparation of rabbit polyclonal antibodies].

Objective To clone, express and purify PPE15 recombinant protein from Mycobacterium tuberculosis (H37Rv), as well as prepare and characterize its rabbit polyclonal antibody. Methods By The PPE15 gene was amplified from the genome of Mycobacterium tuberculosis H37Rv by PCR, and the His-tagged prokaryotic PPE15 prokaryotic expression plasmid pET28a-PPE15 was constructed by homologous recombination cloning technique, and transformed into E. coli BL21 (DE3). PPE15 expression was induced by isopropyl-ß-D-thiogalactopyranoside (IPTG). Recombinant PPE15 was identified by SDS-PAGE, and further purified by affinity chromatography with a Ni-NTA column. The renaturation purified PPE15 protein was used to immunize New-Zealand rabbit to prepare polyclonal antibodies. The antibody specificity was analyzed by Western blot analysis, and antibody titer was determined by indirect ELISA. Results Recombinant prokaryotic PPE15 protein was successfully expressed and purified with a molecular weight of 38 kDa. The purified PPE15 protein exhibited positive reaction with the serum of TB patients and the PPE15 protein, the titer of the polyclonal antibodies reaches more than 1:1 300 480. Conclusion The recombinant protein PPE15 was successfully expressed and purified, and high titer rabbit-derived polyclonal antibody was prepared which provided an experimental basis for further functional studies of PPE15 protein.

Epstein-Barr Virus Induces Lymphangiogenesis and Lympth Node Metastasis via Upregulation of VEGF-C in Nasopharyngeal Carcinoma.

Lymphatic metastasis is a common clinical symptom in nasopharyngeal carcinoma (NPC), the most common Epstein-Barr virus (EBV)-associated head and neck malignancy. However, the effect of EBV on NPC lymph node (LN) metastasis is still unclear. In this study, we demonstrated that EBV infection is strongly associated with advanced clinical N stage and lymphangiogenesis of NPC. We found that NPC cells infected with EBV promote LN metastasis by inducing cancer-associated lymphangiogenesis, whereas these changes were abolished upon clearance of EBV genomes. Mechanistically, EBV-induced VEGF-C contributed to lymphangiogenesis and LN metastasis, and PHLPP1, a target of miR-BART15, partially contributed to AKT/HIF1a hyperactivity and subsequent VEGF-C transcriptional activation. In addition, administration of anti-VEGF-C antibody or HIF1α inhibitors attenuated the lymphangiogenesis and LN metastasis induced by EBV. Finally, we verified the clinical significance of this prometastatic EBV/VEGF-C axis by determining the expression of PHLPP1, AKT, HIF1a, and VEGF-C in NPC specimens with and without EBV. These results uncover a reasonable mechanism for the EBV-modulated LN metastasis microenvironment in NPC, indicating that EBV is a potential therapeutic target for NPC with lymphatic metastasis. IMPLICATIONS: This research demonstrates that EBV induces lymphangiogenesis in NPC by regulating PHLPP1/p-AKT/HIF1a/VEGF-C, providing a new therapeutic target for NPC with lymphatic metastasis.

Codon Optimization, Soluble Expression and Purification of PE_PGRS45 Gene from Mycobacterium tuberculosis and Preparation of Its Polyclonal Antibody Protein.

Studies have demonstrated that PE_PGRS45 is constitutively expressed under various environmental conditions (such as nutrient depletion, hypoxia, and low pH) of the in vitro growth conditions examined, indicating that PE_PGRS45 protein is critical to the basic functions of Mycobacterium tuberculosis. However, there are few reports about the biochemical function and pathogenic mechanism of PE_PGRS45 protein. The fact that this M. tuberculosis gene is not easily expressed in E. coli may be mainly due to the high content of G+C and the use of unique codons. Fusion tags are indispensable tools used to improve the soluble expression of recombinant proteins and accelerate the characterization of protein structure and function. In the present study, His6, Trx, and His6-MBP were used as fusion tags, but only MBP-PE_PGRS45 was expressed solubly. The purification using His6-MBP tag-specific binding to the Ni column was easy to separate after the tag cleavage. We used the purified PE_PGRS45 to immunize New Zealand rabbits and obtained anti- PE_PGRS45 serum. We found that the titer of polyclonal antibodies against PE_PGR45 was higher than 1:256000. The result shows that purified PE_PGRS45 can induce New Zealand rabbits to produce high-titer antibodies. In conclusion, the recombinant protein PE_PGRS45 was successfully expressed in E. coli and specific antiserum was prepared, which will be followed by further evaluation of these specific antigens to develop highly sensitive and specific diagnostic tests for tuberculosis.

Corrigendum: Exploring the Mechanism of Total Flavonoids of Drynariae Rhizoma to Improve Large Bone Defects by Network Pharmacology and Experimental Assessment.

[This corrects the article DOI: 10.3389/fphar.2021.603734.].