Paradoxical Mitophagy Regulation by PINK1 and TUFm.

Aberrant mitophagy has been implicated in a broad spectrum of disorders. PINK1, Parkin, and ubiquitin have pivotal roles in priming mitophagy. However, the entire regulatory landscape and the precise control mechanisms of mitophagy remain to be elucidated. Here, we uncover fundamental mitophagy regulation involving PINK1 and a non-canonical role of the mitochondrial Tu translation elongation factor (TUFm). The mitochondrion-cytosol dual-localized TUFm interacts with PINK1 biochemically and genetically, which is an evolutionarily conserved Parkin-independent route toward mitophagy. A PINK1-dependent TUFm phosphoswitch at Ser222 determines conversion from activating to suppressing mitophagy. PINK1 modulates differential translocation of TUFm because p-S222-TUFm is restricted predominantly to the cytosol, where it inhibits mitophagy by impeding Atg5-Atg12 formation. The self-antagonizing feature of PINK1/TUFm is critical for the robustness of mitophagy regulation, achieved by the unique kinetic parameters of p-S222-TUFm, p-S65-ubiquitin, and their common kinase PINK1. Our findings provide new mechanistic insights into mitophagy and mitophagy-associated disorders.

Pepsinogen ratio and brachial-ankle pulse wave velocity: a cross-sectional study on their interrelationship in atherosclerosis.

BACKGROUND: Existing research has established the pepsinogen ratio (PGR) as a complex biomarker, not only as an independent predictor for various gastrointestinal diseases but also in its association with atherosclerotic cardiovascular diseases. However, the precise mechanism linking changes in PGR to cardiovascular pathologies remains unclear. The objective of this study is to quantitatively elucidate the association between PGR and brachial-ankle pulse wave velocity (baPWV) as an indicator of atherosclerotic progression. METHODS: We conducted a cross-sectional study that analyzed clinical data from 465 patients who underwent health screenings. One-way Analysis of Variance (ANOVA) identified potential risk factors affecting baPWV. Multiple logistic regression was employed to evaluate if PGR serves as an independent risk factor for elevated baPWV after accounting for these variables. Generalized additive models and smoothed curve fitting were utilized to investigate the possibility of a nonlinear association between PGR and baPWV. When such nonlinearity was found, threshold effect analysis pinpointed the inflection point in this relationship, followed by segmented correlation analyses. RESULTS: PGR negatively correlated with both right baPWV (RbaPWV) and left baPWV (LbaPWV) after adjusting for confounders. Smoothed curve analyses revealed nonlinear relationships, with inflection points at 22.5 for RbaPWV and 22.3 for LbaPWV. For PGR values below 22.5, a significant negative correlation with RbaPWV was observed (ß = - 6.3 cm/s, P < 0.001). Conversely, for PGR values above 22.5, no significant linear relationship was found (P = 0.141). Similarly, when PGR was below 22.3, a strong negative correlation with LbaPWV was detected (ß = - 7.0 cm/s, P < 0.001), but such correlation was absent for higher PGR levels (P = 0.273). CONCLUSION: The study reveals that PGR is associated with RbaPWV and LbaPWV in a nonlinear manner. Specifically, lower levels of PGR were linearly and inversely correlated with baPWV, but this relationship became nonlinear at higher PGR levels. These findings suggest that modulating PGR levels may offer a therapeutic strategy for managing atherosclerosis.

ß-Catenin-treated peptides effectively inhibit the proliferation of colorectal cancer.

To verify the inhibitory mechanism of ß-catenin-designed peptides in colorectal cancer(CRC) tumors, the following experiments were performed. In vitro colony formation, Transwell assays, and flow cytometry were performed to assess the biological effects of designed peptides (F18KD, F20A4-7k, F20A4-10k, and F20A3-9k + F20A4-10k + F20A5-9k) in HT-29 cells. In vivo xenograft experiments were performed and treated with peptides. Next, tumors were subjected to Hematoxylin and eosin staining (HE), immunohistochemical, and terminal deoxynucleotidyl transferase dUTP nick end labeling staining assays to evaluate the inhibitory effect of peptides on tumors. ß-Catenin levels were quantified via western blotting (WB) and quantitative real-time polymerase chain reaction, and ß-catenin was located using confocal laser scanning microscopy. T-cell factor-4 (TCF-4), C-myc, and CCND1 levels were quantified via WB. Results were obtained as following. First, the peptides reduced viability, migration, and invasion; promoted apoptosis; and stabilized the S phase of HT-29 cells. Second, peptides suppressed tumor growth and downregulated the expression of CD34, vascular endothelial growth factor, and ß-catenin in tumors. Furthermore, we found that peptides downregulated ß-catenin expression in both the cytoplasm and nucleus; TCF-4, C-myc, and CCND1 expression was also downregulated. Notably, ß-catenin-targeting peptides had a better inhibitory effect on CRC than non-ß-catenin-target peptides, and a combination of peptides exerted a more potent inhibitory effect on CRC than single peptides. It suggested that ß-Catenin-targeting peptides promote apoptosis in CRC tumors by inhibiting activation of the Wnt/ß-catenin pathway.

Monocyte to high-density lipoprotein cholesterol ratio and serum uric acid in Chinese adults: a cross-sectional study.

BACKGROUND: Previous studies have shown that the monocyte to high-density lipoprotein cholesterol (HDL-C) ratio (MHR) is a predictor of various diseases such as coronary heart disease, diabetic microangiopathy, and metabolic syndrome. However, there are few scientific reports on the correlation between MHR and serum uric acid. The objective of this report is to explore the relationship between MHR and serum uric acid in Chinese adults. METHODS: This cross-sectional study included 646 participants from southwest China who underwent a health examination at the Health Management Center of Deyang People's Hospital. The examination included blood pressure readings, routine blood tests (lipid, fasting glucose, serum transaminase, and serum uric acid levels), and various standardized questionnaires. We employed a generalized additive model and smoothed curve fitting to explore the relationship between MHR and serum uric acid levels. We then performed subgroup analyses to investigate the robustness of this relationship. RESULTS: After adjusting for confounders (age, sex, body mass index, systolic blood pressure, diastolic blood pressure, aspartate transaminase, alanine aminotransferase, fasting glucose, total cholesterol, low-density lipoprotein, smoking, drinking, and exercise status), MHR was found to be positively correlated with serum uric acid levels (P < 0.001). The smoothing curve showed an approximately linear correlation between MHR and serum uric acid levels, and the linear correlation coefficient was 146.74 (95% CI 96.16-197.33, P < 0.0001). The subgroup analyses showed that the effect of MHR on serum uric acid levels was smaller in occasional smokers and smokers than in nonsmokers (P = 0.0194). CONCLUSION: MHR was significantly and positively correlated with serum uric acid levels. Additionally, the effect of MHR on serum uric acid levels was lower in the individuals who smoked more.

Overexpression and purification of HSV-2 glycoprotein D in suspension CHO cells with serum-free medium and immunogenicity analysis.

Glycoprotein D (gD2) is the most important candidate antigen for herpes simplex virus type 2 (HSV-2) vaccine development. Establishment of a stable eukaryotic cell line to overexpress gD2 and an efficient purification process to purify is essential for the development of subunit vaccine against HSV-2. The DNA sequence of the extracellular epitope-rich fragment of gD2 was optimized, chemically synthesized, and cloned into plasmid pMD902. The recombinant plasmid pMD902-gD was stably transfected into CHO-DG44 cells, and cell lines with high levels of expression of gD2 were established. The recombinant gD2 was purified efficiently using an anion exchange column and a Sephadex G-25 desalting column. The yield of the purified gD2 was 57 mg/L of serum-free culture medium, and its purity was determined to be about 95% by HPLC analysis. Finally, the immunogenicity of the purified gD2 was measured and it induced strong and specific humoral immunity and higher level of cellular immune response than gD2 expressed in prokaryotic cells. We established a stable, secretory, and high-yield gD2-expression cell line and an easy and efficient gD2-purification process, which lays the foundation for preparation of large amount of gD2 that is essential for HSV-2 subunit vaccine development.

Machine Learning for Prediction of Non-Small Cell Lung Cancer Based on Inflammatory and Nutritional Indicators in Adults: A Cross-Sectional Study.

Purpose: The aim of this study was to evaluate the potential benefit of blood inflammation in the diagnosis of non-small cell lung cancer (NSCLC) and propose a machine-learning-based method to predict NSCLC in asymptomatic adults. Patients and Methods: A cross-sectional study was evaluated using medical records of 139 patients with non-small cell lung cancer and physical examination data from May 2022 to May 2023 of 198 healthy controls. The NSCLC cohort comprised 128 cases of adenocarcinoma, 3 cases of squamous cell carcinoma, and 8 cases of other NSCLC subtypes. The correlation between inflammatory and nutritional markers, such as monocytes, neutrophils, LMR, NLR, PLR, PHR and non-small cell lung cancer was examined. Features were selected using Python's feature selection library and analyzed by five algorithms. The predictive ability of the model for non-small cell lung cancer diagnosis was assessed by precision, accuracy, recall, F1 score, and area under the curve (AUC). Results: The results showed that the top 14 important factors were PDW, age, TP, RBC, HGB, LYM, LYM%, RDW, PLR, LMR, PHR, MONO, MONO%, gender. Additionally, the naive Bayes (NB) algorithm demonstrated the highest overall performance in predicting adult NSCLC among the five machine learning algorithms, achieving an accuracy of 0.87, a macro average F1 score of 0.85, a weighted average F1 score of 0.87, and an AUC of 0.84. Conclusion: In feature ranking, platelet distribution width was the most important feature, and the NB algorithm performed best in predicting adult NSCLC diagnosis.

Time series models in prediction of severe fever with thrombocytopenia syndrome cases in Shandong province, China.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by the SFTS virus (SFTSV). Predicting the incidence of this disease in advance is crucial for policymakers to develop prevention and control strategies. In this study, we utilized historical incidence data of SFTS (2013-2020) in Shandong Province, China to establish three univariate prediction models based on two time-series forecasting algorithms Autoregressive Integrated Moving Average (ARIMA) and Prophet, as well as a special type of recurrent neural network Long Short-Term Memory (LSTM) algorithm. We then evaluated and compared the performance of these models. All three models demonstrated good predictive capabilities for SFTS cases, with the predicted results closely aligning with the actual cases. Among the models, the LSTM model exhibited the best fitting and prediction performance. It achieved the lowest values for mean absolute error (MAE), mean square error (MSE), and root mean square error (RMSE). The number of SFTS cases in the subsequent 5 years in this area were also generated using this model. The LSTM model, being simple and practical, provides valuable information and data for assessing the potential risk of SFTS in advance. This information is crucial for the development of early warning systems and the formulation of effective prevention and control measures for SFTS.

Potential novel Colpodella spp. (phylum Apicomplexa) and high prevalence of Colpodella spp. in goat-attached Haemaphysalis longicornis ticks in Shandong province, China.

Tick-borne Apicomplexan parasites pose a significant threat to both public health and animal husbandry. Identifying potential pathogenic parasites and gathering their epidemiological data are essential for prospectively preventing and controlling infections. In the present study, genomic DNA of ticks collected from two goat flocks (Goatflock1 and Goatflock2) and one dog group (Doggroup) were extracted and the 18S rRNA gene of Babesia/Theileria/Colpodella spp. was amplified by PCR and sequenced. Phylogenetic analysis was conducted based on the obtained sequences. The differences in pathogen positive rates between ticks of different groups were statistically analyzed using the Chi-square or continuity-adjusted Chi-square test. As a result, two pathogenic Theileria (T.) luwenshuni genotypes, one novel pathogenic Colpodella sp. HLJ genotype, and two potential novel Colpodella spp. (referred to as Colpodella sp. struthionis and Colpodella sp. yiyuansis in this study) were identified in the Haemaphysalis (H.) longicornis ticks. Ticks of Goatflock2 had a significantly higher positive rate of Colpodella spp. than those from Goatflock1 (χ2=92.10; P = 8.2 × 10-22) and Doggroup (χ2=42.34; P = 7.7 × 10-11), and a significantly higher positive rate of T. luwenshuni than Doggroup (χ2=5.38; P = 0.02). However, the positive rates of T. luwenshuni between Goatflock1 and Goatflock2 were not significantly different (χ2=2.02; P = 0.16), and so as the positive rates of both pathogens between Goatflock1 and Doggroup groups (P > 0.05). For either Colpodella spp. or T. luwenshuni, no significant difference was found in prevalence between male and female ticks. These findings underscore the potential importance of Colpodella spp. in domestic animal-attached ticks, as our study revealed two novel Colpodella spp. and identified Colpodella spp. in H. longicornis for the first time. The study also sheds light on goats' potential roles in the transmission of Colpodella spp. to ticks and provides crucial epidemiological data of pathogenic Theileria and Colpodella. These data may help physicians, veterinarians, and public health officers prepare suitable detection and treatment methods and develop prevention and control strategies.

Prediction and identification of potential immunodominant epitopes in glycoproteins B, C, E, G, and I of herpes simplex virus type 2.

Twenty B candidate epitopes of glycoproteins B (gB2), C (gC2), E (gE2), G (gG2), and I (gI2) of herpes simplex virus type 2 (HSV-2) were predicted using DNAstar, Biosun, and Antheprot methods combined with the polynomial method. Subsequently, the biological functions of the peptides were tested via experiments in vitro. Among the 20 epitope peptides, 17 could react with the antisera to the corresponding parent proteins in the EIA tests. In particular, five peptides, namely, gB2(466-473) (EQDRKPRN), gC2(216-223) (GRTDRPSA), gE2(483-491) (DPPERPDSP), gG2(572-579) (EPPDDDDS), and gI2(286-295) (CRRRYRRPRG) had strong reaction with the antisera. All conjugates of the five peptides with the carrier protein BSA could stimulate mice into producing antibodies. The antisera to these peptides reacted strongly with the corresponding parent glycoproteins during the Western Blot tests, and the peptides reacted strongly with the antibodies against the parent glycoproteins during the EIA tests. The antisera against the five peptides could neutralize HSV-2 infection in vitro, which has not been reported until now. These results suggest that the immunodominant epitopes screened using software algorithms may be used for virus diagnosis and vaccine design against HSV-2.

BCG Vaccination: A potential tool against COVID-19 and COVID-19-like Black Swan incidents.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19), and its variants have brought unprecedented impacts to the global public health system, politics, economy, and other fields. Although more than ten COVID-19 specific vaccines have been approved for emergency use, COVID-19 prevention and control still face many challenges. Bacille Calmette-Guérin (BCG) is the only authorized vaccine used to fight against tuberculosis (TB), it has been hypothesized that BCG may prevent and control COVID-19 based on BCG-induced nonspecific immune responses. Herein, we summarized: 1) The nonspecific protection effects of BCG, such as prophylactic protection effects of BCG on nonmycobacterial infections, immunotherapy effects of BCG vaccine, and enhancement effect of BCG vaccine on unrelated vaccines; 2) Recent evidence of BCG's efficacy against SARS-COV-2 infection from ecological studies, analytical analyses, clinical trials, and animal studies; 3) Three possible mechanisms of BCG vaccine and their effects on COVID-19 control including heterologous immunity, trained immunity, and anti-inflammatory effect. We hope that this review will encourage more scientists to investigate further BCG induced non-specific immune responses and explore their mechanisms, which could be a potential tool for addressing the COVID-19 pandemic and COVID-19-like "Black Swan" events to reduce the impacts of infectious disease outbreaks on public health, politics, and economy.

CRISPR-Based Diagnostics: A Potential Tool to Address the Diagnostic Challenges of Tuberculosis.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis, which infects more than 23% of the world's population. With the emergence of drug-resistant TB (DR-TB) and latent TB infection (LTBI), rapid diagnosis of DR-TB and LTBI has become a challenge for the prevention and control of TB. Herein, we highlight these challenges and discuss emerging clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics in TB detection. Currently, the clinical diagnosis of M. tuberculosis infection mainly depends on pathogenic and molecular biological methods, including sputum smear, sputum culture, and Xpert. Although CRISPR-based diagnostics have not been applied to the clinical diagnosis of TB, they have shown exciting preponderances in TB diagnosis compared with traditional methods, including higher sensitivity, less sample input, and shorter turnaround time. CRISPR-based diagnostics represent a potential tool to address the challenges and natural weaknesses associated with traditional TB diagnosis methods. Based on the currently available data, we suggest that future CRISPR-based TB diagnostics should be developed in the direction of automation, modularization, diversification, and intelligence. By combining the CRISPR platform with various systems, such as microfluidic chips, droplet microfluidics, electrochemical techniques, and optical systems, the specificity and sensitivity of TB diagnosis may be revolutionized.

A multi-epitopes tandem antigen for five types of human adenoviruses and its application in development of multivalent IgM immunochromatographic strip test.

In China, human adenovirus serotype 3 (HAdV-3), HAdV-7, HAdV-11, HAdV-14, and HAdV-55 are the main prevalent serotypes causing severe acute respiratory diseases and even deaths. To develop multivalent vaccine and diagnostic reagent, a multi-epitopes tandem antigen (META) was designed. Recombinant META was prepared and its humoral immunogenicity, inducing neutralization antibody ability, antigenicity, and reactogenicity were evaluated. A multivalent immunochromatographic strip constructed using the rMETA was evaluated for its sensitivity and specificity in detecting specific IgM antibodies. As a result, the rMETA induced high titers of specific IgG antibodies, with limited abilities of neutralizing multiple HAdVs. It performed both strong antigenicity and reactogenicity. The multivalent immunochromatographic strip recognized specific IgM antibodies against all the 5 types with sensitivities of 87.5% to 95.3%. It performed high specificity of 97.8%. The present study provides both novel idea for developing multivalent vaccine and reagent for point-of-care detection of multiple types of HAdVs.

High prevalence of Rickettsia spp. in ticks from wild hedgehogs rather than domestic bovine in Jiangsu province, Eastern China.

Background: Spotted fever group Rickettsia (SFGR), containing various pathogenic Rickettsia spp., poses remarkable negative influences to public health by causing various severe or mild diseases. Information regarding prevalence of SFGR in ticks in Jiangsu province, Eastern China, is still limited and needs urgent investigations. Methods: Hedgehog- and bovine-attached ticks were collected from Jiangsu province, Eastern China. DNA of individual ticks was extracted for nested polymerase chain reaction amplifications targeting gltA, 16S ribosomal RNA (rrs), ompA, ompB, and sca4 genes following with sequencing. SFGR-specific IgG antibodies in sera of local donators were evaluated using ELISA. Results: Overall, 144 (83.2%) of the 173 ticks from hedgehogs and 2 (1.2%) of the 168 ticks from bovine were positive for one of the three identified Rickettsia spp., with significant difference between the two groups (P = 3.6e-52). Candidatus Rickettsia principis (9; 5.2%) and R. heilongjiangensis (135; 78.0%) were detected in Haemaphysalis flava rather than in H. longicornis ticks from hedgehogs. R. heilongjiangensis (1; 0.6%) and Candidatus R. jingxinensis (or Candidatus R. longicornii) (1; 0.6%) were identified in H. longicornis and Rhipicephalus microplus ticks from bovine, respectively. Phylogenetic analysis indicated Candidatus R. jingxinensis belonged to R. japonica subgroup, whereas Candidatus R. principis belonged to a novel subgroup. Higher serological prevalence of spotted fever and SFGR-specific IgG antibody level in humans were observed around the investigated area than in urban areas, without significant difference. Conclusion: Candidatus R. principis and Candidatus R. jingxinensis were identified in Jiangsu province, Eastern China, and fully genetically characterized for the first time. The higher prevalence of SFGR in hedgehog-attached ticks as well as the higher SFGR-specific IgG antibody level and seropositive rate in humans around the investigated area suggested that more attention should be paid to SFGR. This pathogen is usually transmitted or harbored by wild animals and ticks. This study provides important epidemiological data for both physicians and public health officers in developing early prevention and control strategies against potential Rickettsia infections and in the preparation of suitable testing and treatment needs for rickettsiosis in the endemic areas.

Design and evaluation of a multi-epitope assembly peptide (MEAP) against herpes simplex virus type 2 infection in BALB/c mice.

BACKGROUND: Human herpes simplex virus (HSV) 1 and 2 causes oral, ocular, or genital infections, which remains a significant health problem worldwide. HSV-1 and -2 infections in humans range from localized skin infections of the oral, ocular, and genital regions to severe and often disseminated infections in immunocompromised hosts. Epitope based vaccination is a promising mean to achieve protective immunity and to avoid infections with Human herpes simplex virus type 2 (HSV-2). METHODS: The twelve selected epitopes, six B cell epitopes from different glycoprotein of HSV-2 (amino acid residues 466-473 (EQDRKPRN) from envelope glycoprotein B, 216-223 (GRTDRPSA) from C, 6-18 (DPSLKMADPNRFR) from D, 483-491 (DPPERPDSP) from E, 572-579 (EPPDDDDS) from G and 286-295 (CRRRYRRPRG) from I glycoprotein of HSV-2), four CD4+ T cell epitopes (amino acid residues 21-28 (NLPVLDQL) from D, 162-177 (KDVTVSQVWFGHRYSQ) from B, 205-224 (KAYQQGVTVDSIGMLPRFIP) from D and 245-259 (KPPYTSTLLPPELSD) from D) and two CD8+ T cell epitopes (amino acid residues 10-20 (KMADPNRFRGK) from D and 268-276 (ALLEDPAGT) from D), are responsible for the elicitation of the neutralizing antibodies and cytotoxic T lymphocytes (CTLs) that impart protective immunity to the host. In this study, all above epitopes were inserted into the extracellular fragment (amino acid residues 1-290) of HSV-2 glycoprotein D to construct multi-epitope assembly peptides (MEAPs) by replacing some non-epitope amino acid sequences. The epitope independency of the MEAPs was predicted by three-dimensional software algorithms. The gene of the selected MEAP was expressed in E.coli BL21(DE3), and its protective efficacy against HSV-2 infection was assessed in BALB/c mice. RESULTS: The MEAP, with each inserted epitopes independently displayed on the molecule surface, was selected as candidate proteins. The results showed that the MEAP was highly immunogenic and could elicit high titer neutralizing antibodies and cell-mediated immune responses. CONCLUSIONS: The MEAP provided complete protection against infection with HSV-2 in mice, which indicates that it might be a potential candidate vaccine against HSV-2.

Development of Rapid and Visual Nucleic Acid Detection Methods towards Four Serotypes of Human Adenovirus Species B Based on RPA-LF Test.

OBJECTIVES: Human adenoviruses (HAdV) are classified as 7 HAdV species, and some serotypes in species B like HAdV 3, HAdV 7, HAdV 21, and HAdV 55 caused severe symptoms, even fatalities. Patients may be misdiagnosed and inadequately treated without reliable and practical methods for HAdV serotyping. Developing rapid, sensitive, and specific diagnostic methods for HAdV is critical. METHODS: Detection methods were established based on a recombinase polymerase amplification (RPA) assay and lateral flow (LF) test. Specific target sequence was screened, targeting which, primers and probes were designed, synthesized, and screened for establishing assay with high amplification efficiency. Primer or probe concentrations and amplification time were optimized. Detection limit, sensitivity, and specificity were evaluated. Results and Conclusions. Simple, sensitive, and specific RPA-LF methods for detection of four serotypes of HAdV together or separately were established, which had detection limits of 10 to 280 copies/reaction comparable to real-time PCR without recognizing other pathogens. The sensitivity and specificity were >92% and >98%, respectively, evaluated by limited clinical samples. The detection can be completed in 25 min without requirement of any instrument except a constant temperature equipment, showing superior detection performance and promising for a wide use in the field and resource-limited area.

Evaluation of the Integrated Information System for Brucellosis Case Diagnosis and Management - Inner Mongolia Autonomous Region, China, 2019.

What is already known about this topic? Wulanchabu City Center for Endemic Disease Prevention and Control had established and used a Brucellosis Integrated Information System (BIIS) since 2013. However, it had not been systematically evaluated and promoted so far. What is added by this report? The BIIS had significantly improved the efficiency of brucellosis reporting and provided convenience for follow-up management of cases, which was valuable for finishing completely routine therapy. However, the stability of the system needs to be improved. What are the implications for public health practice? The results of the BIIS assessment demonstrated its advantages and disadvantages, which could provide some evidence for its implementation in other areas of China.

Identification of a Ribosomal Protein RpsB as a Surface-Exposed Protein and Adhesin of Rickettsia heilongjiangensis.

Rickettsia heilongjiangensis is an obligate intracellular bacterium that is responsible for far-eastern spotted fever. Surface-exposed proteins (SEPs) play important roles in its pathogenesis. Previous work identified a ribosomal protein RpsB as an SEP by biotin-avidin affinity, a seroreactive antigen, and a diagnostic candidate protein, indicating that it might play an important role in the pathogenesis of rickettsiae. However, in the absence of other evidence, its subcellular location of being surface-exposed was puzzling because ribosomal proteins are located in the cytoplasm. In the present study, the subcellular location of RpsB was analyzed with bioinformatics tools coupled with immunoelectron microscopy. The adhesion ability of RpsB was evaluated by protein microarray and cellular ELISA. Consequently, different bioinformatics tools gave different location predication results. Thus, RpsB was found in the cytoplasma and inner and outer membranes of R. heilongjiangensis by transmission electron microscopy. Protein microarray and cellular ELISA showed that RpsB binds to the host cell surface and its adhesion ability was even stronger than the known adhesin Adr1. In conclusion, RpsB was visually and directly shown for the time to be an SEP of rickettsiae and might be an important ligand and adhesin of rickettsiae. Its roles in pathogenesis warrant further study.

Development of a rapid and visual detection method for Rickettsia rickettsii combining recombinase polymerase assay with lateral flow test.

OBJECTIVES: Rickettsia rickettsii is the causative agent of Rocky Mountain spotted fever, which is the most severe spotted fever group (SFG) rickettsiosis. Developing a simple and reliable detection method is required. METHODS: A detection method for R. rickettsii was established based on a recombinase polymerase amplification (RPA) assay and the lateral flow (LF) test. A specific target sequence was screened, and corresponding primers and probes were designed, synthesized, and screened for establishing an RPA assay with high amplification efficiency. Reagent concentrations, amplification time, and loading volume for strip development were optimized. The detection limit, analytic sensitivity and specificity were evaluated. RESULTS: A rapid, visual, sensitive and specific method for the detection of R. rickettsii based on RPA and the LF test was successfully established. The novel method had a limit of detection of 10 to 50 copies/reaction without recognizing other organisms. Analytical sensitivity and specificity were ≥90% and 100%, respectively, as evaluated by animal and simulative human samples. CONCLUSIONS: Using the established method, detection could be completed in 30 min with visually detectable results by the naked eye, without requirement of any instrument except a constant temperature equipment. The technique shows superior detection performance and is promising for wide use in the field as well as resource-limited areas for R. rickettsii detection.

Development of a rapid and visual nucleotide detection method for a Chinese epidemic strain of Orientia tsutsugamushi based on recombinase polymerase amplification assay and lateral flow test.

OBJECTIVES: Orientia tsutsugamushi is an obligate intracellular pathogen that causes scrub typhus. Diagnosing scrub typhus remains a challenge, and a sensitive, specific, simple, and rapid diagnostic test is still needed. METHODS: A recombinase polymerase amplification (RPA) assay combined with a lateral flow (LF) test targeting the 56-kDa gene of a Karp-like strain of O. tsutsugamushi was developed and optimized. The detection limits, sensitivity, specificity, and simulative clinical performance were evaluated. RESULTS: Primers and probe were screened to establish the RPA assay, and the reaction conditions were optimized. The detection limit was 10 copies/reaction in detecting plasmid DNA and 12 copies/reaction in detecting genomic DNA. The RPA-LF method could differentiate O. tsutsugamushi from other phylogenetically related bacteria. The sensitivity was 100% and specificity was over 90%, when evaluated using infected animal samples or simulative clinical samples. Furthermore, the method was completed in 20min at 37°C followed by a 3-5min incubation at room temperature for the development of an immunochromatographic strip, and the results could be determined visually. CONCLUSIONS: This method is promising for wide-ranging use in basic medical units considering that it requires minimal instruments and infrastructure and is highly time-efficient, sensitive, and specific for diagnosing scrub typhus.