Clinical Efficacy of Programmed Cell Death Ligand 1 Antibody in Treatment of Extranodal Natural Killer/T-Cell Lymphoma With Hemophagocytic Lymphohistiocytosis.

Extranodal natural killer/T-cell lymphoma-associated hemophagocytic lymphohistiocytosis (ENKTCL-LAHS) is a rare disease with poor prognosis. Currently, there are no well-established treatments for LAHS. Almost 50% of patients experience relapsed or refractory disease to anti-hemophagocytic lymphohistiocytosis (HLH) treatment, and the regimen for salvage therapy is limited. We report a case of ENKTCL-LAHS that was successfully treated with a programmed cell death ligand 1 (PD-L1) antibody (sugemalimab) alone and provide a literature review on existing ENKTCL-LAHS treatment options. A 31-year-old man with relapsed ENKTCL complicated by HLH was admitted to our hospital. Following the administration of the PD-L1 antibody sugemalimab, fever was resolved, Epstein-Barr virus (EBV) DNA copy number was negative, and HLH-related blood biochemical markers were decreased in the patient. Consequently, the patient achieved complete remission with a progression-free time (PFS) of 44 months. The prognosis of ENKTCL-LAHS is extremely poor, and the clinical treatment of ENKTCL-HLH is challenging. No previous reports exist regarding the use of PD-L1 antibodies in ENKTCL-LAHS treatment. This study is the first to report a patient with ENKTCL-LAHS treated with the PD-L1 antibody alone, who achieved a long PFS of 44 months. Our results suggest the effectiveness and safety of sugemalimab in the treatment of ENKTCL-LAHS; however, more clinical cases are required for validation. The PD-L1 antibody presents a novel treatment option for patients with ENKTCL-LAHS and warrants further clinical promotion.

The protective effects of BMSA1 and BMSA5-1-1 proteins against Babesia microti infection.

The intracellular parasite Babesia microti is among the most significant species causing human babesiosis and is an emerging threat to human health worldwide. Unravelling the pathogenic molecular mechanisms of babesiosis is crucial in developing new diagnostic and preventive methods. This study assessed how priming with B. microti surface antigen 1 (BHSA 1) and seroreactive antigen 5-1-1 (BHSA 5-1-1) mediate protection against B. microti infection. The results showed that 500 µg/ml rBMSA1 and rBMSA5-1-1 partially inhibited the invasion of B. microti in vitro by 42.0 ± 3.0%, and 48.0 ± 2.1%, respectively. Blood smears revealed that peak infection at 7 days post-infection (dpi) was 19.6%, 24.7%, and 46.7% in the rBMSA1, rBmSA5-1-1, compared to the control groups (healthy mice infected with B. microti only), respectively. Routine blood tests showed higher white blood cell, red blood cell counts, and haemoglobin levels in the 2 groups (BMSA1 and BMSA5 5-1-1) than in the infection control group at 0-28 dpi. Moreover, the 2 groups had higher serum interferon-γ, tumor necrosis factor-α and Interleukin-17A levels, and lower IL-10 levels than the infection control group throughout the study. These 2 potential vaccine candidate proteins partially inhibit in vitro and in vivo B. microti infection and enhance host immunological response against B. microti infection.

Self-reported sleep characteristics are linked to type 2 diabetes in middle-aged and elderly individuals: a cross-sectional study based on NHANES.

OBJECTIVE: This study was aimed to evaluate the link between sleep characteristics and type 2 diabetes of middle-aged and elderly individuals. METHODS: Twenty thousand four hundred ninety-seven individuals enrolled in National Health and Nutritional Examination Survey (NHANES) form periods of 2005-2008 were included in this study, and 3965 individuals aged 45 years and older with complete data were detected. Variables related to sleep characteristics were analyzed by univariate analysis to identify the risk factors of type 2 diabetes, the logistic regression model was used to test for the tendency across the sections of sleep duration, and the link between sleep duration and risk of type 2 diabetes was manifested as odds ratio (OR) and 95% confidence interval (CI). RESULTS: Six hundred ninety-four individuals with type 2 diabetes were identified and enrolled in the type 2 diabetes group, while the remaining individuals (n = 3271) were enrolled in the non-type 2 diabetes group. Individuals in the type 2 diabetes group (63.9 ± 10.2) were older than those in the non-type 2 diabetes group (61.2 ± 11.5, P < 0.001). Factors of taking longer time to fall asleep (P < 0.001), sleeping less (≤ 4 h) or more (≥ 9 h) (P < 0.001), having trouble in falling asleep (P = 0.001), frequent snoring (P < 0.001), frequent sleep apnea (P < 0.001), frequent nighttime awakenings (P = 0.004), and frequent excessive daytime sleepiness (P < 0.001) were linked to the risk of type 2 diabetes. CONCLUSION: Our study revealed that sleep characteristics were closely linked to type 2 diabetes in middle-aged and elderly individuals, and a longer sleep duration might have protective effects against type 2 diabetes, but it should be constrained within 9 h/night.

Clinical evaluation of prone position ventilation in the treatment of acute respiratory distress syndrome induced by sepsis.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is an acute, diffuse, inflammatory lung injury. Previous studies have shown prone position ventilation (PPV) to be associated with improvement in oxygenation. However, its role in patients with ARDS caused by sepsis remains unknown. AIM: To analyze the clinical effects of PPV in patients with ARDS caused by sepsis. METHODS: One hundred and two patients with ARDS were identified and divided into a control group (n = 55) and a PPV treatment group (n = 47). Outcomes included oxygenation index, lung compliance (Cst) and platform pressure (Pplat), which were compared between the two groups after ventilation. Other outcomes included heart rate (HR), mean arterial pressure (MAP), central venous pressure (CVP), left ventricular ejection fraction (LVEF), the length of mechanical ventilation time and intensive care unit (ICU) stay, and levels of C-reactive protein (CRP), procalcitonin (PCT), and interleukin-6 (IL-6) after ventilation. Finally, mortality rate was also compared between the two groups. RESULTS: On the first day after ventilation, the oxygenation index and Cst were higher and Pplat level was lower in the PPV group than in the conventional treatment group (P < 0.05). There were no significant differences in oxygenation index, Cst, and Pplat levels between the two groups on the 2nd, 4th, and 7th day after ventilation (P > 0.05). There were no significant differences in HR, MAP, CVP, LVEF, duration of mechanical ventilation and ICU stay, and the levels of CRP, PCT, and IL-6 between the two groups on the first day after ventilation (all P > 0.05). The mortality rates on days 28 and 90 in the PPV and control groups were 12.77% and 29.09%, and 25.53% and 45.45%, respectively (P < 0.05). CONCLUSION: PPV may improve respiratory mechanics indices and may also have mortality benefit in patients with ARDS caused by sepsis. Finally, PPV was not shown to cause any adverse effects on hemodynamics and inflammation indices.

Modulating the Fluorescence of Silver Nanoclusters Wrapped in DNA Hairpin Loops via Confined Strand Displacement and Transient Concatenate Ligation for Amplifiable Biosensing.

It is intriguing to modulate the fluorescence emission of DNA-scaffolded silver nanoclusters (AgNCs) via confined strand displacement and transient concatenate ligation for amplifiable biosensing of a DNA segment related to SARS-CoV-2 (s2DNA). Herein, three stem-loop structural hairpins for signaling, recognizing, and assisting are designed to assemble a variant three-way DNA device (3WDD) with the aid of two linkers, in which orange-emitting AgNC (oAgNC) is stably clustered and populated in the closed loop of a hairpin reporter. The presence of s2DNA initiates the toehold-mediated strand displacement that is confined in this 3WDD for repeatable recycling amplification, outputting numerous hybrid DNA-duplex conformers that are implemented for a transient "head-tail-head" tandem ligation one by one. As a result, the oAgNC-hosted hairpin loops are quickly opened in loose coil motifs, bringing a significant fluorescence decay of multiple clusters dependent on s2DNA. Demonstrations and understanding of the tunable spectral performance of a hairpin loop-wrapped AgNC via switching 3WDD conformation would be highly beneficial to open a new avenue for applicable biosensing, bioanalysis, or clinical diagnostics.

Target Deoxyribonucleic Acid-Recycled Lighting-Up Amplifiable Ratiometric Fluorescence Biosensing of Bicolor Silver Nanoclusters Hosted in a Switchable Deoxyribonucleic Acid Construct.

Ratiometric assays of label-free dual-signaling reporters with enzyme-free amplification are intriguing yet challenging. Herein, yellow- and red-silver nanocluster (yH-AgNC and rH-AgNC) acting as bicolor ratiometric emitters are guided to site-specifically cluster in two template signaling hairpins (yH and rH), respectively, and originally, both of them are almost non-fluorescent. The predesigned complement tethered in yH is recognizable to a DNA trigger (TOC) related to SARS-CoV-2. With the help of an enhancer strand (G15E) tethering G-rich bases (G15) and a linker strand (LS), a switchable DNA construct is assembled via their complementary hybridizing with yH and rH, in which the harbored yH-AgNC close to G15 is lighted-up. Upon introducing TOC, its affinity ligating with yH is further implemented to unfold rH and induce the DNA construct switching into closed conformation, causing TOC-repeatable recycling amplification through competitive strand displacement. Consequently, the harbored rH-AgNC is also placed adjacent to G15 for turning on its red fluorescence, while the yH-AgNC is retainable. As demonstrated, the intensity ratio dependent on varying TOC is reliable with high sensitivity down to 0.27 pM. By lighting-up dual-cluster emitters using one G15 enhancer, it would be promising to exploit a simpler ratiometric biosensing format for bioassays or clinical theranostics.

Antibody-Responsive Ratiometric Fluorescence Biosensing of Biemissive Silver Nanoclusters Wrapped in Switchable DNA Tweezers.

Exploring the ratiometric fluorescence biosensing of DNA-templated biemissive silver nanoclusters (AgNCs) is significant in bioanalysis, yet the design of a stimuli-responsive DNA device is a challenge. Herein, using the anti-digoxin antibody (anti-Dig) with two identical binding sites as a model, a tweezer-like DNA architecture is assembled to populate fluorescent green- and red-AgNCs (g-AgNCs and r-AgNCs), aiming to produce a ratio signal via specific recognition of anti-Dig with two haptens (DigH). To this end, four DNA probes are programmed, including a reporter strand (RS) dually ended with a g-/r-AgNC template sequence, an enhancer strand (ES) tethering two same G-rich tails (G18), a capture strand (CS) labeled with DigH at two ends, and a help strand (HS). Initially, both g-AgNCs and r-AgNCs wrapped in the intact RS are nonfluorescent, whereas the base pairing between RS, ES, CS, and HS resulted in the construction of DNA mechanical tweezers with two symmetric arms hinged by a rigid "fulcrum", in which g-AgNCs are lighted up due to G18 proximity ("green-on"), and r-AgNCs away from G18 are still dark ("red-off"). When two DigHs in proximity recognize and bind anti-Dig, the conformation switch of these tweezers resultantly occurs, taking g-AgNCs away from G18 for "green-off" and bringing r-AgNCs close to G18 for "red-on". As such, the ratiometric fluorescence of r-AgNCs versus g-AgNCs is generated in response to anti-Dig, achieving reliable quantization with a limit of detection at the picomolar level. Based on the fast stimulated switch of unique DNA tweezers, our ratiometric strategy of dual-emitting AgNCs would provide a new avenue for a variety of bioassays.

Symptomatic and Asymptomatic Protist Infections in Hospital Inpatients in Southwestern China.

Cryptosporidium spp., Entamoeba histolytica, Giardia duodenalis, and Blastocystis sp. infections have been frequently reported as etiological agents for gastroenteritis, but also as common gut inhabitants in apparently healthy individuals. Between July 2016 and March 2017, stool samples (n = 507) were collected from randomly selected individuals (male/female ratio: 1.1, age range: 38-63 years) from two sentinel hospitals in Tengchong City Yunnan Province, China. Molecular (PCR and Sanger sequencing) methods were used to detect and genotype the investigated protist species. Carriage/infection rates were: Blastocystis sp. 9.5% (95% CI: 7.1-12.4%), G. duodenalis 2.2% (95% CI: 1.1-3.8%); and E. histolytica 2.0% (95% CI: 0.9-3.6%). Cryptosporidium spp. was not detected at all. Overall, 12.4% (95% CI: 9.7-15.6) of the participants harbored at least one enteric protist species. The most common coinfection was E. histolytica and Blastocystis sp. (1.0%; 95% CI: 0.3-2.2). Sequence analyses revealed that 90.9% (10/11) of the genotyped G. duodenalis isolates corresponded to the sub-assemblage AI. The remaining sequence (9.1%, 1/11) was identified as sub-assemblage BIV. Five different Blastocystis subtypes, including ST3 (43.7%, 21/48), ST1 (27.1%, 13/48), ST7 (18.8%, 9/48), ST4 (8.3%, 4/48), and ST2 (2.1%, 1/48) were identified. Statistical analyses confirmed that (i) the co-occurrence of protist infections was purely random, (ii) no associations were observed among the four protist species found, and (iii) neither their presence, individually or jointly, nor the patient's age was predictors for developing clinical symptoms associated with these infections. Overall, these protist mono- or coinfections are asymptomatic and do not follow any pattern.

Molecular Characterization and Immunological Evaluation of Truncated Babesia microti Rhoptry Neck Protein 2 as a Vaccine Candidate.

Babesia microti is a protozoan that infects red blood cells. Babesiosis is becoming a new global threat impacting human health. Rhoptry neck proteins (RONs) are proteins located at the neck of the rhoptry and studies indicate that these proteins play an important role in the process of red blood cell invasion. In the present study, we report on the bioinformatic analysis, cloning, and recombinant gene expression of two truncated rhoptry neck proteins 2 (BmRON2), as well as their potential for incorporation in a candidate vaccine for babesiosis. Western blot and immunofluorescence antibody (IFA) assays were performed to detect the presence of specific antibodies against BmRON2 in infected mice and the localization of N-BmRON2 in B. microti parasites. In vitro experiments were carried out to investigate the role of BmRON2 proteins during the B. microti invasion process and in vivo experiments to investigate immunoprotection. Homologous sequence alignment and molecular phylogenetic analysis indicated that BmRON2 showed similarities with RON2 proteins of other Babesia species. We expressed the truncated N-terminal (33-336 aa, designated rN-BmRON2) and C-terminal (915-1171 aa, designated rC-BmRON2) fragments of the BmRON2 protein, with molecular weights of 70 and 29 kDa, respectively. Western blot assays showed that the native BmRON2 protein is approximately 170 kDa, and that rN-BmRON2 was recognized by serum of mice experimentally infected with B. microti. Immunofluorescence analysis indicated that the BmRON2 protein was located at the apical end of merozoites, at the opposite end of the nucleus. In vitro red blood cell invasion inhibition studies with B. microti rBmRON2 proteins showed that relative invasion rate of rN-BmRON2 and rC-BmRON2 group is 45 and 56%, respectively. Analysis of the host immune response after immunization and B. microti infection showed that both rN-BmRON2 and rC-BmRON2 enhanced the immune response, but that rN-BmRON2 conferred better protection than rC-BmRON2. In conclusion, our results indicate that truncated rhoptry neck protein 2, especially its N-terminal fragment (rN-BmRON2), plays an important role in the invasion of host red blood cells, confers immune protection, and shows good potential as a candidate vaccine against babesiosis.

Efficacy of the combination of modern medicine and traditional Chinese medicine in pulmonary fibrosis arising as a sequelae in convalescent COVID-19 patients: a randomized multicenter trial.

BACKGROUND: The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to a significant number of mortalities worldwide. COVID-19 poses a serious threat to human life. The clinical manifestations of COVID-19 are diverse and severe and 20% of infected patients are reported to be in a critical condition. A loss in lung function and pulmonary fibrosis are the main manifestations of patients with the severe form of the disease. The lung function is affected, even after recovery, thereby greatly affecting the psychology and well-being of patients, and significantly reducing their quality of life. METHODS: Participants must meet the following simultaneous inclusion criteria: over 18 years of age, should have recovered from severe or critical COVID-19 cases, should exhibit pulmonary fibrosis after recovery, and should exhibit Qi-Yin deficiency syndrome as indicated in the system of traditional Chinese medicine (TCM). The eligible candidates will be randomized into treatment or control groups. The treatment group will receive modern medicine (pirfenidone) plus TCM whereas the control group will be administered modern medicine plus TCM placebo. The lung function index will be continuously surveyed and recorded. By comparing the treatment effect between the two groups, the study intend to explore whether TCM can improve the effectiveness of modern medicine in patients with pulmonary fibrosis arising as a sequelae after SARS-CoV-2 infection. DISCUSSION: Pulmonary fibrosis is one of fatal sequelae for some severe or critical COVID-19 cases, some studies reveal that pirfenidone lead to a delay in the decline of forced expiratory vital capacity, thereby reducing the mortality partly. Additionally, although TCM has been proven to be efficacious in treating pulmonary fibrosis, its role in treating pulmonary fibrosis related COVID-19 has not been explored. Hence, a multicenter, parallel-group, randomized controlled, interventional, prospective clinical trial has been designed and will be conducted to determine if a new comprehensive treatment for pulmonary fibrosis related to COVID-19 is feasible and if it can improve the quality of life of patients. TRIAL REGISTRATION: This multicenter, parallel-group, randomized controlled, interventional, prospective trial was registered at the Chinese Clinical Trial Registry (ChiCTR2000033284) on 26th May 2020 (prospective registered).

Quietness of circular RNA circ_0054633 alleviates the inflammation and proliferation in lipopolysaccharides-induced acute lung injury model through NF-κB signaling pathway.

AIM: Acute lung injury (ALI) is the mild form of acute respiratory distress syndrome (ARDS) which is a common lung disease with a high incidence and mortality rate. Recent studies manifested that some circular RNAs were associated with ALI. In this study, we aimed to uncover the effect of circular RNA circ_0054633 on ALI initiation and progression and proposed a new mechanism related to ALI. METHODS: The lipopolysaccharides (LPS)-induced acute lung injury model were build both in vivo of rat and in vitro of primary murine pulmonary microvascular endothelial cells (MPVECs). Hematoxylin and eosin (H&E) was employed to observe the tissue morphology and estimate the degree of lung damage. We used real-time quantitative polymerase chain reaction (RT-qPCR) to measure the expression level of circ_0054633. The expression levels of inflammatory cytokines IL-17A and tumor necrosis factor-α (TNF-α) were detected by ELISA. The effects of circ_0054633 on MPVECs proliferation and apoptosis were detected with the help of CCK-8 and apoptosis assay, separately. The expression level of NF-κB p65 protein was measured by Western blot. RESULTS: circ_0054633, IL-17A, TNF-α and NF-κB p65 were all overexpressed in LPS-treated rat and MPVECs, and LPS enhanced the proliferation and apoptosis of MPVECs. While circ_0054633 silencing reversed the above promotion effects of LPS on IL-17A, TNF-α expression and MPVECs proliferation and apoptosis. CONCLUSIONS: Quietness of circ_0054633 alleviated LPS-induced ALI via NF-κB signaling pathway, implicating circ_0054633 may be a potential biomarker for diagnose and therapy of ALI.

Microwave hyperthermia enhances the sensitivity of lung cancer cells to gemcitabine through reactive oxygen species­induced autophagic death.

The pleiotropic effects of hyperthermia on cancer cells have been well documented, and microwave hyperthermia (MWHT) has been widely applied for multifarious cancer treatment. However, the mechanisms underlying the anticancer effect of MWHT combined with gemcitabine (GEM) remain poorly understood. The aim of the present study was to investigate the role of autophagy in the thermo­chemotherapy of human squamous cell lung carcinoma cells. It was observed that MWHT combined with GEM potently suppressed the viability of NCI­H2170 and NCI­H1703 cells, and induced G0/G1 cell cycle arrest. Notably, MWHT with GEM induced autophagy, as indicated by the formation of autophagic vacuoles, downregulation of p62 and upregulation of light chain 3­II. It was further demonstrated that the autophagy was due to the production of reactive oxygen species (ROS), whereas N­acetyl cysteine, an ROS scavenger, attenuated the level of autophagy. However, when the autophagy inhibitor 3­methyladenine was used, there was no significant change in the production of ROS. Furthermore, it was observed that MWHT combined with GEM downregulated the protein expression levels of phosphoinositide 3­kinase (PI3K), phosphorylated (p)­PI3K, protein kinase B (AKT), p­AKT, mammalian target of rapamycin (mTOR), p­mTOR, phosphorylated S6 (pS6) and p70 S6 kinase, which are associated with autophagy. In addition, the results demonstrated that ROS served as an upstream mediator of PI3K/AKT/mTOR signaling. In light of these findings, the present study provides original insights into the molecular mechanisms underlying the cell death induced by MWHT combined with GEM, and this may be a promising approach for the treatment of human squamous cell lung carcinoma.

Small-scale spatial analysis of intermediate and definitive hosts of Angiostrongylus cantonensis.

BACKGROUND: Angiostrongyliasis is a food-borne parasitic zoonosis. Human infection is caused by infection with the third-stage larvae of Angiostrongylus cantonensis. The life cycle of A. cantonensis involves rodents as definitive hosts and molluscs as intermediate hosts. This study aims to investigate on the infection status and characteristics of spatial distribution of these hosts, which are key components in the strategy for the prevention and control of angiostrongyliasis. METHODS: Three villages from Nanao Island, Guangdong Province, China, were chosen as study area by stratified random sampling. The density and natural infection of Pomacea canaliculata and various rat species were surveyed every three months from December 2015 to September 2016, with spatial correlations of the positive P. canaliculata and the infection rates analysed by ArcGIS, scan statistics, ordinary least squares (OLS) and geographically weighted regression (GWR) models. RESULTS: A total of 2192 P. canaliculata specimens were collected from the field, of which 1190 were randomly chosen to be examined for third-stage larvae of A. cantonensis. Seventy-two Angiostrongylus-infected snails were found, which represents a larval infection rate of 6.1% (72/1190). In total, 110 rats including 85 Rattus norvegicus, 10 R. flavipectus, one R. losea and 14 Suncus murinus were captured, and 32 individuals were positive (for adult worms), representing an infection rate of 29.1% of the definitive hosts (32/110). Worms were only found in R. norvegicus and R. flavipectus, representing a prevalence of 36.5% (31/85) and 10% (1/10), respectively in these species, but none in R. losea and S. murinus, despite testing as many as 32 of the latter species. Statistically, spatial correlation and spatial clusters in the spatial distribution of positive P. canaliculata and positive rats existed. Most of the spatial variability of the host infection rates came from spatial autocorrelation. Nine spatial clusters with respect to positive P. canaliculata were identified, but only two correlated to infection rates. The results show that corrected Akaike information criterion, R2, R2 adjusted and σ2 in the GWR model were superior to those in the OLS model. CONCLUSIONS: P. canaliculata and rats were widely distributed in Nanao Island and positive infection has also been found in the hosts, demonstrating that there was a risk of angiostrongyliasis in this region of China. The distribution of positive P. canaliculata and rats exhibited spatial correlation, and the GWR model had advantage over the OLS model in the spatial analysis of hosts of A. cantonensis.

[Chemical constituents from roots of Datura metel].

The chemical constituents from the n-butanol fraction of the 70% ethanol extract of Datura metel roots were separated by silica gel and ODS chromatogram columns as well as preparative HPLC. On the basis of spectral data analysis, their structures were elucidated. Twenty-one compounds were obtained and their structures were identified as citroside A (1), coniferin (2), paeoniflorin (3), (6R,7E,9R)-9-hydroxy-4,7-megastigmadien-3-one 9-O-[α-L-arabin-opyranosyl-(l→6)-ß-D-glucopyranoside] (4), (1R,7R,10R,11R)-12-hydroxyl anhuienosol (5), kaurane acid glycoside A (6), ent-2-oxo-15,16-dihydroxypimar-8(14)-en-16-O-ß-glucopyranoside (7), ginsenoside Rg1(8), ginsenoside Re (9), notoginsenosides R1(10), N-butyl-O-ß-D-fructofuranoside (11), salidroside (12), hexyl ß-sophoroside (13), 2,6-dimethoxy-4-hydroxyphenol 1-glucoside (14), benzyl-O-ß-D-xylopyranoxyl(1→6)-ß-D-glucopyranoside (15), (Z)-3-hexenyl-O-α-L-arabinopyranosyl-(1→6)-ß-D-glucopyranoside (16), N-[2-(3,4-dihydro-xyphenyl)-2-hydroxyethyl]-3-(4-methoxyphenyl) prop-2-enamide (17), cannabisin D (18), cannabisin E (19), melongenamide B (20), paprazine (21). Compounds 2-17 and 20-21 were isolated from the Solanaceae family for the first time.

Molecular characterization of Babesia microti seroreactive antigen 5-1-1 and development of rapid detection methods for anti-B. microti antibodies in serum.

Babesiosis has become a new global threat impacting human health, and most human babesiosis cases are caused by Babesia microti. Until now few antigens of B. microti have been described which can be used for the diagnosis of human babesiosis. In the present study, we report on the bioinformatic analysis, cloning and expression of the sequence encoding the B. microti seroreactive antigen 5-1-1 to investigate its potential incorporation in serologic diagnostic tools for babesiosis. Bioinformatic analysis and recombinant gene expression were performed to molecularly characterize seroreactive antigen 5-1-1. Enhanced chemiluminescence (ECL)-Western blot methods were used to detect specific antibodies in infected mice. Immunofluorescence antibody assays (IFA) were performed to detect the localization of BmSA5-1-1 in B. microti parasites. ELISA and immunochromatographic (ICT) tests were developed using recombinant BmSA5-1-1 to evaluate its potential use in rapid detection methods for B. microti antibodies and for the diagnosis of babesiosis. A recombinant expression plasmid was constructed by inserting the target gene fragment in the pET28a vector after double digestion with BamHI and XhoI restriction enzymes. The recombinant BmSA5-1-1 protein was expressed in Escherichia coli (rBmSA5-1-1) and purified by means of Ni-nitrilotriacetic acid (NTA) agarose columns. Polyclonal antibodies were generated against rBmSA5-1-1. Based on indirect immunofluorescence assay results, BmSA5-1-1 appeared to localize on the surface of B. microti. ELISA tests using the rBmSA5-1-1 antigen detected specific antibodies from infected mice as early as 4 days post-infection. Our results indicate that the two methods we developed can detect specific antibodies in mice at different stages of infection with sensitivities of 100% (rBmSA5-1-1 ELISA) and 90% (ICT). The specificity of the two methods was 100%. Sera of patients suffering from other closely related parasitic diseases, such as malaria and toxoplasmosis, produced negative results. In conclusion, seroreactive antigen 5-1-1, a member of the BMN1 protein family, is expressed on the outer surface of B. microti and is a promising candidate antigen for the early diagnosis of babesiosis. rBmSA5-1-1 ELISA and ICT methods show good potential for detecting specific antibodies in mice at different stages of infection.

Behavioral study of a rat model of migraine induced by CGRP.

Migraine is a debilitating disorder characterized by recurrent headache arising from neurovascular dysfunction. Despite recent progress in migraine research, the exact mechanisms underpinning migraine are poorly understood. Furthermore, it is difficult to develop an animal model of migraine that resembles all symptoms of patients. In this study, we established a novel animal model of migraine induced by epidural injection of calcitonin gene-related peptide (CGRP), and examined climbing hutch behavior, facial-grooming behavior, body-grooming behavior, freezing behavior, resting behavior, and ipsilateral hindpaw facial grooming behavior of rats following CGRP injection. CGRP significantly reduced climbing hutch behavior, and face-grooming behavior, and increased immobile behavior. We also found that the P15 and P85 percentile range of behavioral data exhibited a high positive rate (83.3%) for establishing the model with less false positive rate. Our results verified that the rat model of migraine induced by CGRP featured many behaviors of migraine patients demonstrated during migraine attacks. Our findings suggest that this new model can be a useful tool for understanding the pathophysiology of migraine and studying novel therapeutic strategies for the treatment of migraine.

Case-control study of diarrheal disease etiology in individuals over 5 years in southwest China.

BACKGROUND: Acute diarrhea is one of the major public health problems worldwide. Most of studies on acute diarrhea have been made on infants aged below 5 years and few efforts have been made to identify the etiological agents of acute diarrhea in people over five, especially in China. METHODS: 271 diarrhea cases and 149 healthy controls over 5 years were recruited from four participating hospitals between June 2014 and July 2015. Each stool specimen was collected to detect a series of enteric pathogens, involving five viruses (Rotavirus group A, RVA; Norovirus, NoV; Sapovirus, SaV; Astrovirus, As; and Adenovirus, Ad), seven bacteria (diarrheagenic Escherichia coli, DEC; non-typhoidal Salmonella, NTS; Shigella spp.; Vibrio cholera; Vibrio parahaemolyticus; Aeromonas spp.; and Plesiomonas spp.) and three protozoa (Cryptosporidium spp., Giardia lamblia, G. lamblia, and Blastocystis hominis, B. hominis). Standard microbiological and molecular methods were applied to detect these pathogens. Data was analyzed using Chi square, Fisher-exact tests and logistic regressions. RESULTS: The prevalence of at least one enteric pathogen was detected in 29.2% (79/271) acute diarrhea cases and in 12.1% (18/149) in healthy controls (p < 0.0001). Enteric viral infections (14.4%) were the most common in patients suffering from acute diarrhea, followed by bacteria (13.7%) and intestinal protozoa (4.8%). DEC (12.5%) was the most common causative agent in diarrhea cases, followed by NoV GII (10.0%), RVA (7.4%) and B. hominis (4.8%). The prevalence of co-infection was statistically higher (p = 0.0059) in the case group (7.7%) than in the healthy control (1.3%). RVA-NoV GII (3.0%) was the most common co-infection in symptomatic cases. CONCLUSIONS: DEC was the most predominant pathogen in diarrhea cases, but it was largely overlooked because the lack of laboratory capacities. Because of the high prevalence of co-infections, it is recommended the urgent development of alternative laboratory methods to assess polymicrobial infections. Such methodological improvements will result in a better prevention and treatment strategies to control diarrhea illness in China.

[The clinical research of nystatin in prevention of invasive fungal infections in patients on mechanical ventilation in intensive care unit].

OBJECTIVE: To observe the effect of nystatin on incidence of invasive fungal infections (IFI) and the prognosis of mechanically ventilated critically ill patients. METHODS: A prospective study was conducted. Critical ill patients admitted to Department of Critical Care Medicine of Jiangxi Provincial People's Hospital from May 1st, 2012 to April 30th, 2013 needing mechanical ventilation were enrolled. The patients were randomly divided into two groups by envelope method. Patients in the nystatin group were administered nystatin 1000 kU three times a day via the gastric tube; and patients in the control group were given gastrointestinal prokinetic drug as placebo. The specimens were collected every 3 days throughout the ICU stay (T0, T3, T6, T9), the strain distribution was observed, and the corrected colonization index (CCI) of all patients were calculated. The incidence of candidemia and 28-day mortality as well as the duration of stay in ICU and hospital were also recorded. RESULTS: A total of 874 strains were isolated from 124 patients, of which Candida albicans accounted for 57.6% (503/874). The most frequently colonized body sites were oropharyngeal site,account for 35.6% (311/874). The CCI of the nystatin group were lower than those of the control group at T6 and T9 [T6: 0.19±0.10 vs. 0.39±0.15, T9: 0.00 (0.10) vs. 0.45 (0.30), all P<0.05]. The incidence of candidemia in the nystatin group was slightly lower than that in control group [0.5% (3/60) vs. 7.8% (5/64), P>0.05]. The mortality in the nystatin group was lower than that in control group [18.3% (11/60) vs. 34.4% (22/64), P<0.05]. ICU day in the nystatin group was shorter than that in the control group (days: 9.56±3.47 vs. 11.89±6.32, P<0.05). However,hospital day was similar in the two groups (days: 18.35±7.42 vs. 20.58±8.77, P>0.05). CONCLUSIONS: Nystatin might reduce the colonization of Candida albicans and was associated with shorter ICU day.

A PTPN22 promoter polymorphism -1123G>C is associated with RA pathogenesis in Chinese.

The minor allele of the non-synonymous single nucleotide polymorphism (SNP) +1858C>T within the PTPN22 gene has now been unequivocally confirmed as conferring susceptibility to RA in population from Europe and America, but not in population from Asia. The aim of this study was to jointly address and integrate these separate findings to further elucidate the association between the PTPN22 gene and RA in Chinese Hans of Guangdong province. Four hundred and ninety-four cases with RA and 496 healthy controls were randomly selected, their SNPs at position -1123G>C (rs2488457), +1858C>T (rs2476601), +788G>A (rs33996649), and rs1310182 were genotyped using PCR-RFLP, followed by agarose gel electrophoresis. +1858C>T (rs2476601) and +788G>A (rs33996649) are not polymorphic in Chinese Hans. Meanwhile, our result reveals that the degree of association between the promoter polymorphism, -1123G>C and RA, was analogous to that observed in Japanese reports (odds ratio [OR] = 1.517, 95% CI = [1.154-1.995], P = 0.003). Expression study also indicated a tendency for association between -1123G>C and PTPN22 gene expression. Our study underpins that the promoter polymorphism, -1123G/C, may be a causal SNP for RA in Asian.

Mutation of tryptophanase gene tnaA in Edwardsiella tarda reduces lipopolysaccharide production, antibiotic resistance and virulence.

A fish pathogen Edwardsiella tarda LTB-4 produced various indole alkaloids, including indole, 2-(1H-indol-3-yl)ethanol, 4-di(1H-indol-3-yl)methylphenol, tri(1H-indol-3-yl)methane and 2-[2,2-bis(1H-indol-3-yl)]ethylphenylamine. Indole was the most abundant among these indole alkaloids. E. tarda LTB-4 produced indoles during its whole growth phase and maintained a high level (around 35.5 µM) during the stationary phase. The relevant tryptophanase (TnaA) gene tnaA was cloned from LTB-4 and conditionally expressed in Escherichia coli; the recombinant TnaA catalysed L-tryptophan to indole. A tnaA in-frame deletion mutant ΔtnaA was constructed through double cross-over allelic exchange by means of the suicide vector pRE118; deletion of tnaA caused some phenotypic changes including decreased swarming and twitching motility, lipopolysaccharide production and multiple antibiotic resistances. Also, subtherapeutic doses of chloromycetin, carbenicillin and tetracyline could cause the decrease of bacterial growth, but greatly induce the production of indole by E. tarda. Most importantly, attenuated virulence of the ΔtnaA mutant to zebra fish by increasing the LD50 for about 55-fold indicated that TnaA involved in the virulence of E. tarda.