Co-infection of human papillomavirus and other sexually transmitted bacteria in cervical cancer patients in the Philippines.

Cervical cancer is the fourth most common cancer in women globally. Based on several epidemiologic studies, human papillomavirus is strongly associated with cervical neoplasia. Aside from HPV, other bacterial infections in the genital tract were associated with cervical neoplasia. This study aimed to determine the prevalence of HPV infection; and co-infection with Ureaplasma spp., Mycoplasma spp., Chlamydia trachomatis, and Neisseria gonorrheae in Filipino cervical cancer patients. Forty-four patients (28 patients with cervical carcinoma and 16 patients with non-malignant cervix) who consulted in the Philippine General Hospital from 2016 to 2017 were included in this study. HPV genotyping and genetic detection of Ureaplasma spp., Mycoplasma spp., C. trachomatis, and N. gonorrheae were done using different PCR assays. The prevalence of HPV 16/18/33/52 was 75% in cervical cancer patients and 25% in control patients. Infection with HPV 16/18/33/52 was significantly associated with having cervical cancer (OR: 9.00; 95% CI: 2.18-37.18; p = 0.0024). HPV-16 was the most prevalent HPV genotype among Filipino cervical cancer patients. HPV-18 and HPV-52 were only detected from cervical cancer patients. Among HPV-positive patients, we noted a 22.73% co-infection with Ureaplasma spp. and 9.09% co-infection with Mycoplasma spp. To our knowledge, this is the first study on the co-infection of HPV and sexually transmitted infections among cervical cancer patients in the Philippines.

Blockade of endoplasmic reticulum stress-induced cell death by Ureaplasma parvum vacuolating factor.

Previously, we found that Ureaplasma parvum internalised into HeLa cells and cytosolic accumulation of galectin-3. U. parvum induced the host cellular membrane damage and survived there. Here, we conducted vesicular trafficking inhibitory screening in yeast to identify U. parvum vacuolating factor (UpVF). U. parvum triggered endoplasmic reticulum (ER) stress and upregulated the unfolded protein response-related factors, including BiP, P-eIF2 and IRE1 in the host cells, but it blocked the induction of the downstream apoptotic factors. MicroRNA library screening of U. parvum-infected cells and UpVF-transfected cells identified miR-211 and miR-214 as the negative regulators of the apoptotic cascade under ER stress. Transient expression of UpVF induced HeLa cell death with intracellular vacuolization; however, some stable UpVF transformant survived. U. parvum-infected cervical cell lines showed resistance to actinomycin D, and UpVF stable transformant cell lines exhibited resistance to X-ray irradiation, as well as cisplatin and paclitaxel. UpVF expressing cervical cancer xenografts in nude mice also acquired resistance to cisplatin and paclitaxel. A mycoplasma expression vector based on Mycoplasma mycoides, Syn-MBA (multiple banded antigen)-UpVF, reduced HeLa cell survival compared with that of Syn-MBA after 72 hr of infection. These findings together suggest novel mechanisms for Ureaplasma infection and the possible implications for cervical cancer malignancy. TAKE AWAYS: • Ureaplasmal novel virulence factor, UpVF, was identified. • UpVF triggered ER stress but suppressed apoptotic cascade via miR-211 and -214. • UpVF conferred resistance to anticancer treatments both in vivo and in vitro. • Dual expression of MBA and UpVF in JCVI-syn3B showed host cell damage.

Development of an efficient one-step real-time reverse transcription polymerase chain reaction method for severe acute respiratory syndrome-coronavirus-2 detection.

The general methods to detect the RNA of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) in clinical diagnostic testing involve reverse transcriptases and thermostable DNA polymerases. In this study, we compared the detection of SARS-CoV-2 by a one-step real-time RT-PCR method using a heat-resistant reverse transcriptase variant MM4 from Moloney murine leukemia virus, two thermostable DNA polymerase variants with reverse transcriptase activity from Thermotoga petrophila K4 and Thermococcus kodakarensis KOD1, or a wild-type DNA polymerase from Thermus thermophilus M1. The highest performance was achieved by combining MM4 with the thermostable DNA polymerase from T. thermophilus M1. These enzymes efficiently amplified specific RNA using uracil-DNA glycosylase (UNG) to remove contamination and human RNase P RNA amplification as an internal control. The standard curve was obtained from 5 to 105 copies of synthetic RNA. The one-step real-time RT-PCR method's sensitivity and specificity were 99.44% and 100%, respectively (n = 213), compared to those of a commercially available diagnostic kit. Therefore, our method will be useful for the accurate detection and quantification of SARS-CoV-2.

Superstructure formation by RodZ hexamers of Shigella sonnei maintains the rod shape of bacilli.

The rod shape of bacilli is maintained by bacterial cytoskeletal protein MreB, an actin homolog that acts in concert with the inner membrane protein RodZ. We previously reported RodZ binds RNA to control the posttranscriptional regulation of invE (virB), which controls the type III secretion system essential for the virulence of Shigella. Here, we show that purified RodZ forms "superstructures" of high molecular mass that dissociate into a midsized "basal complex" in the presence of nonionic detergent, or to a monomer in the presence of dithiothreitol. We used mass spectrometry to show that the basal complex was a hexamer. Electrophoresis mobility shift assays combined with gel filtration detected the RNA-binding activity in fractions containing molecules larger than the basal hexamer. The superstructure was consistently detected with MreB in crude cell lysates of S. sonnei that were fractionated using gel filtration. Immunofluorescence microscopy using two different super-resolution settings showed that wild-type RodZ was distributed in cells as separate dots. Consistent with the superstructure comprising homohexamers, majority of the dots distributed among areas of discrete values. In addition, simultaneous immunodetection of MreB provided the first evidence of colocalization with RodZ as larger patch like signals. These findings indicate that native RodZ forms clusters of various sizes, which may correspond to a superstructure comprising multiple hexamers required for the RNA-binding activity.

Intracellular fate of Ureaplasma parvum entrapped by host cellular autophagy.

Genital mycoplasmas, including Ureaplasma spp., are among the smallest human pathogenic bacteria and are associated with preterm birth. Electron microscopic observation of U. parvum showed that these prokaryotes have a regular, spherical shape with a mean diameter of 146 nm. U. parvum was internalized into HeLa cells by clathrin-mediated endocytosis and survived for at least 14 days around the perinuclear region. Intracellular U. parvum reached endosomes in HeLa cells labeled with EEA1, Rab7, and LAMP-1 within 1 to 3 hr. After 3 hr of infection, U. parvum induced the cytosolic accumulation of galectin-3 and was subsequently entrapped by the autophagy marker LC3. However, when using atg7-/- MEF cells, autophagy was inadequate for the complete elimination of U. parvum in HeLa cells. U. parvum also colocalized with the recycling endosome marker Rab11. Furthermore, the exosomes purified from infected HeLa cell culture medium included U. parvum. In these purified exosomes ureaplasma lipoprotein multiple banded antigen, host cellular annexin A2, CD9, and CD63 were detected. This research has successfully shown that Ureaplasma spp. utilize the host cellular membrane compartments possibly to evade the host immune system.

Human thioredoxin-1 attenuates the rate of lipopolysaccharide-induced preterm delivery in mice in association with its anti-inflammatory effect.

BACKGROUND: Maternal intrauterine infection/inflammation represents the major etiology of preterm delivery and the leading cause of neonatal mortality and morbidity. The aim of this study was to investigate the anti-inflammatory properties of thioredoxin-1 in vivo and its potential ability to attenuate the rate of inflammation-induced preterm delivery. METHODS: Two intraperitoneal injections of lipopolysaccharide from Escherichia coli were administered in pregnant mice on gestational day 15, with a 3-h interval between the injections. From either 1 h before or 1 h after the first lipopolysaccharide injection, mice received three intravenous injections of either recombinant human thioredoxin-1, ovalbumin, or vehicle, with a 3-h interval between injections. RESULTS: Intraperitoneal injection of lipopolysaccharide induced a rise of tumor necrosis factor-α, interferon-γ, monocyte chemotactic protein 1, and interleukin-6 in maternal serum levels and provoked preterm delivery. Recombinant human thoredoxin-1 prevented the rise in these proinflammatory cytokine levels. After the inflammatory challenge, placentas exhibited severe maternal vascular dilatation and congestion and a marked decidual neutrophil activation. These placental pathological findings were ameliorated by recombinant human thioredoxin-1, and the rate of inflammation-induced preterm delivery was attenuated. CONCLUSION: Thioredoxin-1 may thus represent a novel effective treatment to delay inflammation-induced preterm delivery.

Surfactant protein-D attenuates the lipopolysaccharide-induced inflammation in human intestinal cells overexpressing toll-like receptor 4.

PURPOSE: Necrotizing enterocolitis (NEC) is a devastating inflammatory disease of preterm infants that may depend on overexpression of toll-like receptor-4 (TLR4) in the immature intestine. Surfactant protein (SP)-D is a member of the collectin family and plays an important role in innate immunity, particularly in the airways. Although SP-D also exists in the intestines, little is known about its function. This study investigated whether SP-D can attenuate the inflammatory response of TLR4-overexpressing embryonal intestinal cells. METHODS: All experimental procedures were performed using the human intestinal cell line INT407 originally derived from human embryonal intestines. Platelet-activating factor (PAF), reported to be elevated in NEC patients, was used to induce TLR4 overexpression in the human embryonal intestinal cell line INT407. TLR4 expression was measured using quantitative real-time PCR. Inflammatory responses to PAF (5 µM), the TLR4 agonist lipopolysaccharide (LPS, 100 ng/ml), PAF + LPS, and PAF + LPS following SP-D pretreatment (20 µg/ml) were assessed by enzyme-linked immunosorbent assay (ELISA) of interleukin-8 (IL-8) release (in pg/ml). RESULTS: Expression of TLR4 mRNA (mean ± SD) was upregulated by PAF (369 % ± 28 %, p < 0.001). Stimulation with PAF + LPS resulted in higher IL-8 release (1959.3 ± 52.3) than control (141.2 ± 12.4), LPS (167.3 ± 65.8), or PAF (1527.2 ± 129.4) treatment (p < 0.05). Release in response to PAF + LPS (1590.1 ± 319.3) was attenuated by SP-D pretreatment (1161.6 ± 131.6; p < 0.05). CONCLUSION: SP-D attenuates LPS-induced IL-8 production in TLR4-overexpressing intestinal cells, suggesting that SP-D may have a protective effect in the development of NEC in preterm infants.

Short-term changes in intracellular ROS localisation after the silver nanoparticles exposure depending on particle size.

Silver nanoparticles (AgNPs) induce the production of reactive oxygen species (ROS) and apoptosis. These effects are enhanced by smaller particles. Using live-cell imaging, we show that AgNPs induced ROS production rapidly in a size-dependent manner after exposure of cells to 70-nm and 1-nm AgNPs (AgNPs-70, AgNPs-1), but not AgNO3. Exposure of cells to 5 µg/mL each of AgNPs-70, AgNPs-1 or AgNO3 for 1 h decreased the cell viability by approximately 40%, 100% and 20%, respectively. ROS were rapidly induced after 5 and 60 min by AgNPs-1 and AgNPs-70, respectively, whereas AgNO3 had no detectable effect. ROS production detected using the reporter dichlorodihydrofluorescein was observed in whole cells and mitochondria 5 and 60 min after exposure to AgNPs-1. The present study is the first, to our knowledge, to report the temporal expression and intracellular localisation of ROS induced by AgNPs.