Proteomic profiling of extracellular vesicles derived from human serum for the discovery of biomarkers in Avascular necrosis.

BACKGROUND: Avascular necrosis (AVN) is a medical condition characterized by the destruction of bone tissue due to a diminished blood supply. When the rate of tissue destruction surpasses the rate of regeneration, effective treatment becomes challenging, leading to escalating pain, arthritis, and bone fragility as the disease advances. A timely diagnosis is imperative to prevent and initiate proactive treatment for osteonecrosis. We explored the potential of differentially expressed proteins in serum-derived extracellular vesicles (EVs) as biomarkers for AVN of the femoral head in humans. We analyzed the genetic material contained in serum-derived exosomes from patients for early diagnosis, treatment, and prognosis of avascular necrosis. METHODS: EVs were isolated from the serum of both patients with AVN and a control group of healthy individuals. Proteomic analyses were conducted to compare the expression patterns of these proteins by proteomic analysis using LC-MS/MS. RESULTS: Our results show that the levels of IGHV3-23, FN1, VWF, FGB, PRG4, FCGBP, and ZSWIM9 were upregulated in the EVs of patients with AVN compared with those of healthy controls. ELISA results showed that VWF and PRG4 were significantly upregulated in the patients with AVN. CONCLUSIONS: These findings suggest that these EV proteins could serve as promising biomarkers for the early detection and diagnosis of AVN. Early diagnosis is paramount for effective treatment, and the identification of new osteonecrosis biomarkers is essential to facilitate swift diagnosis and proactive intervention. Our study provides novel insights into the identification of AVN-related biomarkers that can enhance clinical management and treatment outcomes.

Analysis of particulate matter-induced alteration of genes and related signaling pathways in the respiratory system.

Airborne particulate matter (PM) is a global environmental risk factor threatening human health and is a major cause of cardiovascular and respiratory disease-associated death. Current studies on PM exposure have been limited to large-scale cohort and epidemiological investigations, emphasizing the need for detailed individual-level studies to uncover specific differentially expressed genes and their associated signaling mechanisms. Herein, we revealed that PM exposure significantly upregulated inflammatory and immune responses, such as cytokine-mediated signaling pathways, complement system, and the activation and migration of immune cells in gene set enrichment analysis of our RNA sequencing (RNAseq) data. Remarkably, we discovered that the broad gene expression and signaling pathways mediated by macrophages were predominantly expressed in the respiratory system following PM exposure. Consistent with these observations, individual PMs, classified by aerodynamic size and origin, significantly promoted macrophage recruitment to the lungs in the mouse lung inflammation model. Additionally, we confirmed that RNAseq observations from the respiratory system were reproduced in murine bone marrow-derived macrophages and the alveolar macrophage cell line MH-S after individual PM exposure. Our findings demonstrated that PM exposure augmented broad inflammatory and immune responses in the respiratory system and suggested the reinforcement of global strategies for reducing particulate air pollution to prevent respiratory diseases and their exacerbation.

Microplastic contamination in groundwater on a volcanic Jeju Island of Korea.

Microplastic (MPs) contamination in groundwater has received massive attention since plastic waste has been released directly into the environment. This study investigates MPs contamination in groundwater on the Jeju volcanic Island, Korea. To the best of our knowledge, this is the first study to identify MPs in groundwater from volcanic islands. A total of 21 sites were sampled for groundwater wells and springs in July and September (2021). Sampling was performed without cross-contamination through quality assurance and quality control. The results showed that MPs abundance ranged from 0.006 to 0.192 particles/L in groundwater samples. Additionally, MPs were detected in deep groundwater wells where the groundwater level was 143 m below ground surface. Eight MPs polymer types, including polypropylene, polyethylene, polyethylene terephthalate, polyvinyl chloride, polystyrene, polyamide, acrylonitrile butadiene styrene, and polyurethane, were detected using Micro-Fourier Transform Infrared Spectroscopy (µ-FT-IR). Most of the detected MPs size ranged from 20 to 100 µm, accounting for 95% of the total. Fragments and fiber shaped MPs were detected, with the majority of them being fragmented in groundwater samples. The concentrations of MPs and major ions in groundwater showed a positive correlation. A negative correlation was observed between MPs concentration and topographic elevation (r = -0.59, p = 0.01). The source of MPs contamination is most likely attributed to agricultural activities, such as plastic mulching and greenhouses, which account for most of the land use in the study area. In this study, MPs entered the aquifer through the soil at the surface and seeped through cracks in fractured rock on basalt with sealed groundwater wells. This study takes 500 L of samples to prevent sample bias, reveal plastic contamination in groundwater, and indicating the characteristics and sources of contaminated plastics.

Extracellular Vesicles from Human Adipose Tissue-Derived Mesenchymal Stem Cells Suppress RANKL-Induced Osteoclast Differentiation via miR122-5p.

Researchers are increasingly interested in cell therapy using mesenchymal stem cells (MSCs) as an alternative remedy for osteoporosis, with fewer side effects. Thus, we isolated and characterized extracellular vesicles (EVs) from human adipose tissue-derived MSCs (hMSCs) and investigated their inhibitory effects on RANKL-induced osteoclast differentiation. Purified EVs were collected from the supernatant of hMSCs by tangential flow filtration. Characterization of EVs included typical evaluation of the size and concentration of EVs by nanoparticle tracking analysis and morphology analysis using transmission electron microscopy. hMSC-EVs inhibited RANKL-induced differentiation of bone marrow-derived macrophages (BMDMs) into osteoclasts in a dose-dependent manner. F-actin ring formation and bone resorption were also reduced by EV treatment of osteoclasts. In addition, EVs decreased RANKL-induced phosphorylation of p38 and JNK and expression of osteoclastogenesis-related genes in BMDMs treated with RANKL. To elucidate which part of the hMSC-EVs plays a role in the inhibition of osteoclast differentiation, we analyzed miRNA profiles in hMSC-EVs. The results showed that has-miR122-5p was present at significantly high read counts. Overexpression of miR122-5p in BMDMs significantly inhibited RANKL-induced osteoclast differentiation and induced defects in F-actin ring formation and bone resorption. Our results also revealed that RANKL-induced phosphorylation of p38 and JNK and osteoclast-specific gene expression was decreased by miR122-5p transfection, which was consistent with the results of hMSC-EVs. These findings suggest that hMSC-EVs containing miR122-5p inhibit RANKL-induced osteoclast differentiation via the downregulation of molecular mechanisms and could be a preventive candidate for destructive bone diseases.

Therapeutic Potential of Mesenchymal Stem Cells (MSCs) and MSC-Derived Extracellular Vesicles for the Treatment of Spinal Cord Injury.

Spinal cord injury (SCI) is a life-threatening condition that leads to permanent disability with partial or complete loss of motor, sensory, and autonomic functions. SCI is usually caused by initial mechanical insult, followed by a cascade of several neuroinflammation and structural changes. For ameliorating the neuroinflammatory cascades, MSC has been regarded as a therapeutic agent. The animal SCI research has demonstrated that MSC can be a valuable therapeutic agent with several growth factors and cytokines that may induce anti-inflammatory and regenerative effects. However, the therapeutic efficacy of MSCs in animal SCI models is inconsistent, and the optimal method of MSCs remains debatable. Moreover, there are several limitations to developing these therapeutic agents for humans. Therefore, identifying novel agents for regenerative medicine is necessary. Extracellular vesicles are a novel source for regenerative medicine; they possess nucleic acids, functional proteins, and bioactive lipids and perform various functions, including damaged tissue repair, immune response regulation, and reduction of inflammation. MSC-derived exosomes have advantages over MSCs, including small dimensions, low immunogenicity, and no need for additional procedures for culture expansion or delivery. Certain studies have demonstrated that MSC-derived extracellular vesicles (EVs), including exosomes, exhibit outstanding chondroprotective and anti-inflammatory effects. Therefore, we reviewed the principles and patho-mechanisms and summarized the research outcomes of MSCs and MSC-derived EVs for SCI, reported to date.

A Randomized, Placebo-Controlled, Respiratory Syncytial Virus Human Challenge Study of the Antiviral Efficacy, Safety, and Pharmacokinetics of RV521, an Inhibitor of the RSV-F Protein.

Effective treatments for respiratory syncytial virus (RSV) infection are lacking. Here, we report a human proof-of-concept study for RV521, a small-molecule antiviral inhibitor of the RSV-F protein. In this randomized, double-blind, placebo-controlled trial, healthy adults were challenged with RSV-A Memphis-37b. After infection was confirmed (or 5 days after challenge virus inoculation), subjects received RV521 (350 mg or 200 mg) or placebo orally every 12 h for 5 days. The primary endpoint was area under the curve (AUC) for viral load, as assessed by reverse transcriptase quantitative PCR (RT-qPCR) of nasal wash samples. The primary efficacy analysis set included subjects successfully infected with RSV who received ≥1 dose of study drug. A total of 66 subjects were enrolled (n = 22 per group); 53 were included in the primary analysis set (RV521 350 mg: n = 16; 200 mg: n = 18; placebo: n = 19). The mean AUC of RT-qPCR-assessed RSV viral load (log10 PFU equivalents [PFUe]/ml · h) was significantly lower with RV521 350 mg (185.26; standard error [SE], 31.17; P = 0.002) and 200 mg (224.35; SE, 37.60; P = 0.007) versus placebo (501.39; SE, 86.57). Disease severity improved with RV521 350 mg and 200 mg versus placebo (P = 0.002 and P = 0.009, respectively, for AUC total symptom score [score × hours]). Daily nasal mucus weight was significantly reduced (P = 0.010 and P = 0.038 for RV521 350 mg and 200 mg, respectively, versus placebo). All treatment-emergent adverse events were grade 1 or 2. No subjects discontinued due to adverse events. There was no evidence of clinically significant viral resistance, and only three variants were detected. RV521 effectively reduced RSV viral load and disease severity in humans and was well tolerated. (This study has been registered at ClinicalTrials.gov under registration no. NCT03258502.).

Maternal and Cord Blood Folate Concentrations Are Inversely Associated with Fetal DNA Hydroxymethylation, but Not DNA Methylation, in a Cohort of Pregnant Canadian Women.

BACKGROUND: Aberrancies in fetal DNA methylation programming may modify disease susceptibility of the offspring. Maternal folate status has potential to alter fetal DNA methylation. OBJECTIVES: We examined the association of maternal and cord blood concentrations of folate and unmetabolized folic acid (UMFA), vitamin B-12, vitamin B-6, and choline with fetal DNA methylation and hydroxymethylation and assessed potential modifying effects of 38 fetal genetic variants in 22 genes. METHODS: Nutrient blood concentrations were measured in 368 pregnant women in early pregnancy (12-16 wk of gestation) and at delivery (37-42 wk of gestation) and in cord blood. DNA methylation and hydroxymethylation in cord blood mononuclear cells were quantified by LC-MS/MS. Pearson partial correlations were used to determine the association between individual nutrients and DNA methylation and hydroxymethylation. RESULTS: Serum and RBC folate and plasma UMFA concentrations (primary outcomes) in early pregnancy, at delivery, and in cord blood were not significantly associated with fetal DNA methylation. In contrast, maternal RBC folate in early pregnancy (r = -0.16, P = 0.04) and cord plasma UMFA (r = -0.23, P = 0.004) were inversely correlated with fetal DNA hydroxymethylation. Neither maternal and cord blood concentrations of other nutrients nor fetal genotypes (secondary outcomes) were significantly associated with fetal DNA methylation or hydroxymethylation. Infants born to mothers with RBC folate concentrations in the highest quartile and serum vitamin B-12 concentrations in the lowest quartile in early pregnancy had significantly lower fetal DNA methylation and higher birth weight compared with those born to mothers with lower RBC folate and higher serum vitamin B-12 concentrations (P = 0.01). CONCLUSIONS: Maternal and cord blood folate concentrations are associated with fetal DNA hydroxymethylation, but not DNA methylation, in a cohort of pregnant Canadian women. The observation that high folate and low vitamin B-12 maternal status in early pregnancy may be associated with decreased fetal DNA methylation and higher birth weight warrants further investigation. This trial was registered at clinicaltrials.gov as NCT02244684.

CX3CR1+ Macrophages and CD8+ T Cells Control Intestinal IgA Production.

Secretory IgA is a key host defense mechanism that controls the intestinal microbiota. We investigated the role of CD11c+CX3CR1+CD64+ macrophages in IgA production in the intestine. Intestinal CX3CR1+ macrophages directly induced IgA secretion by B cells. Ag delivery to lamina propria (LP) CX3CR1+ macrophages specifically induced intestinal IgA production. The induction of IgA by CX3CR1+ macrophages required BAFF, a proliferation-inducing ligand, and TNF-α, but was surprisingly independent of TLR-mediated microbial recognition and retinoic acid signaling. IgA secretion by CX3CR1+ macrophages was enhanced by LP CD8+ T cells through the secretion of IL-9 and IL-13. CX3CR1+ macrophages and CD8+ T cells induced IgA production by B cells independently of mesenteric lymph nodes and Peyer patches. Our data reveal a previously unrecognized cellular circuitry in which LP CX3CR1+ macrophages, B cells, and CD8+ T cells coordinate the protective Ig secretion in the small intestine upon peripheral Ag delivery.

Folic acid supplement use and breast cancer risk in BRCA1 and BRCA2 mutation carriers: a case-control study.

PURPOSE: Supplemental folic acid (the more bioavailable and synthetic form of folate) and breast cancer risk in BRCA mutation carriers have not been studied. We evaluated folic acid, vitamin B6 and vitamin B12 supplement use, and breast cancer risk among BRCA mutation carriers. METHODS: In this case-control study, dietary supplement use was collected from BRCA mutation carriers living in Canada. Supplement use was categorized as never or ever use. Total average daily supplement use was categorized as never, moderate, and high use based on tertiles. Unconditional logistic regression was used to estimate the odds ratio (OR) and 95% confidence intervals (CI) for supplement use and breast cancer risk. RESULTS: We included 129 breast cancer cases and 271 controls. Women who used any folic acid-containing supplement had a significantly decreased risk of breast cancer compared to women who never used a folic acid-containing supplement (OR 0.45; 95%CI 0.25, 0.79; P = 0.006). This was significant for BRCA1 mutation carriers only. The OR for moderate folic acid supplement intake was 0.39; P = 0.01, and high intake was 0.54; P = 0.09, compared to never users. Moderate vitamin B12 supplement intake was associated with decreased risk of breast cancer compared to never use (OR 0.48; 95%CI 0.24, 0.96; P = 0.04). CONCLUSIONS: In this first investigation of folic acid supplement use and breast cancer risk in BRCA mutation carriers, these findings suggest that moderate folic acid- and vitamin B12-containing supplement use may be protective for BRCA-associated breast cancer, particularly among BRCA1 mutation carriers. Future studies with larger samples and prospective follow-up are needed.

Foveation-based reduced resolution driving scheme for immersive virtual reality displays.

This paper introduces a foveation-based driving scheme that can extend line times for high-resolution and wide viewing angle displays in virtual reality applications. In the proposed one, a panel receives the substantially reduced vertical resolution image with consideration of human visual system based on the distance from the foveation point, which also leads to the dramatic data bandwidth reduction. Then, the foveated-rendering image of the full resolution is recovered in the panel by means of multi-output driving shift registers that can apply the same gate pulses to multiple lines. These multiple lines are simultaneously charged with one same line data and multiple resolutions can be presented automatically on a high resolution display. It is verified that effective numbers of lines are reduced to 30.3% and 21.0% for 4,800 × 4,800 and 9,600 × 9,600 resolutions, respectively. Consequently, line times can be extended to 330.0% and 476.2%. In addition, the subjective evaluation has ensured that the foveation-based driving scheme is applicable to high resolution and wide viewing angle displays without any perceivable degradation of image qualities.

Deep-learning-based pixel compensation algorithm for local dimming liquid crystal displays of quantum-dot backlights.

Local dimming techniques have been widely studied to achieve a high contrast ratio and low power consumption for liquid crystal displays. The luminance of a backlight is reduced according to some characteristics of an input image and the pixel data are boosted to compensate for the dimmed backlight. In addition, because a backlight block is affected by adjacent ones, the pixel compensation algorithm requires huge processing power as well as many iterations along with the overall luminance profile information of a backlight. However, a proposed deep-learning-based local dimming algorithm generates the compensated image directly from an input image without any information of backlight's dimming levels. The proposed compensation network is constructed on the basis of the U-net to maintain the high-resolution features in the up-sampling paths through skip-connections. In addition, it is also ensured that the bi-linear interpolation can be used without visible image quality degradation for the reduction on the number of parameters. The proposed networks are trained and verified on a DIV2K 2K image dataset.

Group B Streptococci Induce Proinflammatory Responses via a Protein Kinase D1-Dependent Pathway.

Group B streptococci (GBS) are one of the leading causes of life-threatening illness in neonates. Proinflammatory responses to GBS mediated through host innate immune receptors play a critical role in the disease manifestation. However, the mechanisms involved in proinflammatory responses against GBS, as well as the contribution of signaling modulators involved in host immune defense, have not been fully elucidated. In the present study, we investigated the role of protein kinase D (PKD)1 in the proinflammatory responses to GBS. We found that both live and antibiotic-killed GBS induce activation of PKD1 through a pathway that is dependent on the TLR signaling adaptor MyD88 and its downstream kinase IL-1R-associated kinase 1, but independent of TNFR-associated factor 6. Our studies using pharmacological PKD inhibitors and PKD1-knockdown macrophages revealed that PKD1 is indispensable for GBS-mediated activation of MAPKs and NF-κB and subsequent expression of proinflammatory mediators. Furthermore, systemic administration of a PKD inhibitor protects d-galactosamine-sensitized mice from shock-mediated death caused by antibiotic-killed GBS. These findings imply that PKD1 plays a critical regulatory role in GBS-induced proinflammatory reactions and sepsis, and inhibition of PKD1 activation together with antibiotic treatment in GBS-infected neonates could be an effective way to control GBS diseases.

Antiviral Activity of Oral JNJ-53718678 in Healthy Adult Volunteers Challenged With Respiratory Syncytial Virus: A Placebo-Controlled Study.

Background: Respiratory syncytial virus (RSV) disease has no effective treatment. JNJ-53718678 is a fusion inhibitor with selective activity against RSV. Methods: After confirmation of RSV infection or 5 days after inoculation with RSV, participants (n = 69) were randomized to JNJ-53718678 75 mg (n = 15), 200 mg (n = 17), 500 mg (n = 18), or placebo (n = 17) orally once daily for 7 days. Antiviral effects were evaluated by assessing RSV RNA viral load (VL) area under the curve (AUC) from baseline (before the first dose) until discharge, time-to-peak VL, duration of viral shedding, clinical symptoms, and quantity of nasal secretions. Results: Mean VL AUC was lower for individuals treated with different doses of JNJ-53718678 versus placebo (203.8-253.8 vs 432.8 log10 PFUe.hour/mL). Also, mean peak VL, time to peak VL, duration of viral shedding, mean overall symptom score, and nasal secretion weight were lower in each JNJ-53718678-treated group versus placebo. No clear exposure-response relationship was observed. Three participants discontinued due to treatment-emergent adverse events of grade 2 and 1 electrocardiogram change (JNJ-53718678 75 mg and 200 mg, respectively) and grade 2 urticaria (placebo). Conclusions: JNJ-53718678 at all 3 doses substantially reduced VL and clinical disease severity, thus establishing clinical proof of concept and the compound's potential as a novel RSV treatment. Clinical trials registration: ClinicalTrials.gov: NCT02387606; EudraCT number: 2014-005041-41.

Regulation and rate enhancement during transcription-coupled DNA repair.

Transcription-coupled DNA repair (TCR) is a subpathway of nucleotide excision repair (NER) that is triggered when RNA polymerase is stalled by DNA damage. Lesions targeted by TCR are repaired more quickly than lesions repaired by the transcription-independent "global" NER pathway, but the mechanism underlying this rate enhancement is not understood. Damage recognition during bacterial NER depends upon UvrA, which binds to the damage and loads UvrB onto the DNA. Bacterial TCR additionally requires the Mfd protein, a DNA translocase that removes the stalled transcription complexes. We have determined the properties of Mfd, UvrA, and UvrB that are required for the elevated rate of repair observed during TCR. We show that TCR and global NER differ in their requirements for damage recognition by UvrA, indicating that Mfd acts at the very earliest stage of the repair process and extending the functional similarities between TCR in bacteria and eukaryotes.

Anti-Photoaging Effects of Low Molecular-Weight Fucoidan on Ultraviolet B-Irradiated Mice.

Ultraviolet (UV) B exposure induces DNA damage and production of reactive oxygen species (ROS), which causes skin photoaging through signaling pathways of inflammation and modulation of extracellular matrix remodeling proteins, collagens, and matrix metalloproteinase (MMP). As low molecular-weight fucoidan (LMF) has potential antioxidant and anti-inflammatory properties, we examined the protective effects of LMF against UVB-induced photoaging. A UVB-irradiated mouse model was topically treated with myricetin or LMF at 2.0, 1.0 and 0.2 mg/cm² (LMF2.0, LMF1.0 and LMF0.2, respectively) once a day for 15 weeks. Wrinkle formation, inflammation, oxidative stress, MMP expression, and apoptosis in the treated regions were compared with those in a distilled water-treated photoaging model (UVB control). LMF treatments, particularly LMF2.0 and LMF1.0, significantly inhibited the wrinkle formation, skin edema, and neutrophil recruitment into the photo-damaged lesions, compared with those in the UVB control. While LMF decreased interleukin (IL)-1ß release, it increased IL-10. The LMF treatment inhibited the oxidative stresses (malondialdehyde and superoxide anion) and enhanced endogenous antioxidants (glutathione). Additionally, LMF reduced the mRNA expression of MMP-1, 9, and 13. The histopathological analyses revealed the anti-photoaging effects of LMF exerted via its antioxidant, anti-apoptotic, and MMP-9-inhibiting effects. These suggest that LMF can be used as a skin-protective remedy for photoaging.

A Mobile Game for Patients With Breast Cancer for Chemotherapy Self-Management and Quality-of-Life Improvement: Randomized Controlled Trial.

BACKGROUND: The application of game-based learning in clinical practice has shown potential advantages in previous studies. However, there have been little efforts to use smartphone-based mobile games in the management of adult patients with cancer. OBJECTIVE: The objective of our study was to evaluate if patient education using a mobile game may increase drug compliance, decrease physical side effects of chemotherapy, and improve psychological status in breast cancer patients. METHODS: A total of 76 patients with metastatic breast cancer who were planned to receive cytotoxic chemotherapy were enrolled in this trial. Study participants were randomly assigned to a mobile game play group (game group, n=36) or a conventional education group (control group, n=40) in a ratio of 1:1. The patients were unblinded and followed prospectively for 3 weeks. Outcome measures included time spent for education, compliance to medication, physical side effects, and psychological side effects including quality of life (QoL). RESULTS: Overall, 72 out of 76 patients completed the study after 3 weeks (95%). The subjects in the game group showed high levels of satisfaction with the app. The time spent playing the mobile game in the game group was longer than that spent for self-education in the control group (mean 22.2, SD 6.1 vs mean 5.5, SD 4.0 minutes a day; P<.001). The mobile game group showed better drug adherence (Korean version of the Medication Adherence Rating Scale; mean 7.6, SD 0.7 vs mean 6.5, SD 0.5; P<.001). The use of the mobile game was associated with lower rates of chemotherapy-related side effects, such as nausea, fatigue, numbness of hand or foot, and hair loss, than the control group. The game group exhibited better QoL during chemotherapy (mean 74.9, SD 3.5 vs mean 72.2, SD 5.3; P=.01). However, there were no significant differences in terms of depression and anxiety scales. CONCLUSIONS: This study suggests the feasibility and potentiality of the use of smartphone mobile games for patients with breast cancer receiving chemotherapy. Education using a mobile game led to better patient education, improved drug compliance, decreased side effects, and better QoL compared with conventional education. Mobile games can be used as easy, fun, and effective measures for patient education and have the potential to improve treatment outcomes. TRIAL REGISTRATION: ClinicalTrials.gov NCT03205969; http://clinicaltrials.gov/ct2/show/NCT03205969 (Archived by WebCite at http://www.webcitation.org/71jfSBOq9).

Suboptimal maternal and cord plasma pyridoxal 5' phosphate concentrations are uncommon in a cohort of Canadian pregnant women and newborn infants.

Vitamin B6 is important in fetal development, but little is known of the vitamin B6 status of pregnant women and newborns in North America and potential modifying factors. This prospective study determined maternal and cord plasma concentrations of pyridoxal 5' phosphate (PLP; an indicator of vitamin B6 status) in a convenience sample of 368 Canadian pregnant women and their newborns. The association of maternal intake of vitamin B6 and fetal genetic variants with cord plasma PLP and homocysteine concentrations was also examined. Dietary and supplemental intakes of vitamin B6 were assessed in early and mid to late pregnancy. PLP concentrations were measured in maternal plasma in early pregnancy and at delivery, and in cord plasma. Six fetal variants of the MTHFR and CßS genes were assessed for their association with cord plasma PLP and homocysteine concentrations. Geometric mean (95% CI) PLP concentrations were 107 (98, 116) nmol/L in early pregnancy and 58 (53, 62) nmol/L at delivery, respectively, and 296 (275, 319) nmol/L in cord blood (p < .0001). During early pregnancy and at delivery, 3.6% and 5.5% of women had plasma PLP concentrations <20 nmol/L, respectively. Ninety eight percent of the women with supplemental B6 intake of at least the recommended dietary allowance had PLP concentrations >20 nmol/L. Fetal genetic variants were not associated with cord PLP and homocysteine concentrations. Vitamin B6 deficiency is uncommon in a cohort of Canadian pregnant women due largely to prevalent vitamin B6 supplement use.

Preclinical Characterization of PC786, an Inhaled Small-Molecule Respiratory Syncytial Virus L Protein Polymerase Inhibitor.

Although respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection in infants and young children, attempts to develop an effective therapy have so far proved unsuccessful. Here we report the preclinical profiles of PC786, a potent nonnucleoside RSV L protein polymerase inhibitor, designed for inhalation treatment of RSV infection. PC786 demonstrated a potent and selective antiviral activity against laboratory-adapted or clinical isolates of RSV-A (50% inhibitory concentration [IC50], <0.09 to 0.71 nM) and RSV-B (IC50, 1.3 to 50.6 nM), which were determined by inhibition of cytopathic effects in HEp-2 cells without causing detectable cytotoxicity. The underlying inhibition of virus replication was confirmed by PCR analysis. The effects of PC786 were largely unaffected by the multiplicity of infection (MOI) and were retained in the face of established RSV replication in a time-of-addition study. Persistent anti-RSV effects of PC786 were also demonstrated in human bronchial epithelial cells. In vivo intranasal once daily dosing with PC786 was able to reduce the virus load to undetectable levels in lung homogenates from RSV-infected mice and cotton rats. Treatment with escalating concentrations identified a dominant mutation in the L protein (Y1631H) in vitro In addition, PC786 potently inhibited RSV RNA-dependent RNA polymerase (RdRp) activity in a cell-free enzyme assay and minigenome assay in HEp-2 cells (IC50, 2.1 and 0.5 nM, respectively). Thus, PC786 was shown to be a potent anti-RSV agent via inhibition of RdRp activity, making topical treatment with this compound a novel potential therapy for the treatment of human RSV infections.

Stalled transcription complexes promote DNA repair at a distance.

Transcription-coupled nucleotide excision repair (TCR) accelerates the removal of noncoding lesions from the template strand of active genes, and hence contributes to genome-wide variations in mutation frequency. Current models for TCR suppose that a lesion must cause RNA polymerase (RNAP) to stall if it is to be a substrate for accelerated repair. We have examined the substrate requirements for TCR using a system in which transcription stalling and damage location can be uncoupled. We show that Mfd-dependent TCR in bacteria involves the formation of a damage search complex that can detect lesions downstream of a stalled RNAP, and that the strand specificity of the accelerated repair pathway is independent of the requirement for a lesion to stall RNAP. We also show that an ops (operon polarity suppressor) transcription pause site, which causes backtracking of RNAP, can promote the repair of downstream lesions when those lesions do not themselves cause the polymerase to stall. Our findings indicate that the transcription-repair coupling factor Mfd, which is an ATP-dependent superfamily 2 helicase that binds to RNAP, continues to translocate along DNA after RNAP has been displaced until a lesion in the template strand is located. The discovery that pause sites can promote the repair of nonstalling lesions suggests that TCR pathways may play a wider role in modulating mutation frequencies in different parts of the genome than has previously been suspected.

Low Serum Vitamin B-12 Concentrations Are Prevalent in a Cohort of Pregnant Canadian Women.

BACKGROUND: Among Canadian women of reproductive age, 5% and 20% have serum vitamin B-12 concentrations indicative of deficiency (<148 pmol/L) and marginal status (148-220 pmol/L), respectively. Given the association between suboptimal vitamin B-12 and adverse pregnancy outcomes, an understanding of vitamin B-12 status during pregnancy, and factors that influence it, is required. OBJECTIVE: This prospective analysis from the PREFORM (PREnatal FOlic acid exposuRe on DNA Methylation in the newborn infant) study investigated 1) vitamin B-12 status in a cohort of Canadian pregnant women and their newborns, 2) the association of maternal dietary vitamin B-12 intake with maternal and cord blood concentrations of vitamin B-12 and its biomarkers, and 3) the association of fetal genetic polymorphisms with cord blood concentrations of vitamin B-12 and its biomarkers. METHODS: In pregnant Canadian women (n = 368; mean ± SD age: 32 ± 5 y), vitamin B-12 intakes were assessed in early (0-16 wk) and mid- to late (23-37 wk) pregnancy. Serum vitamin B-12 and plasma total homocysteine (tHcy) and methylmalonic acid (MMA) in maternal blood at 12-16 wk of pregnancy and at delivery (28-42 wk) and in cord blood were measured and compared by using regression analyses. The associations of 28 fetal genetic variants in vitamin B-12 metabolism and cord blood vitamin B-12, tHcy, and MMA concentrations were assessed by using regression analysis, with adjustment for multiple testing. RESULTS: A total of 17% and 38% of women had deficient and 35% and 43% had marginal serum vitamin B-12 concentrations at 12-16 wk of pregnancy and at delivery, respectively. Only 1.9-5.3% had elevated MMA (>271 nmol/L), and no women had elevated tHcy (>13 µmol/L). Maternal dietary vitamin B-12 intake during pregnancy was either weakly associated or not associated with maternal and cord blood vitamin B-12 (r(2) = 0.17-0.24, P < 0.0008), tHcy (P = NS) and MMA (r(2) = 0.05-0.11, P < 0.001). Fetal genetic polymorphisms were not associated with cord blood concentrations of vitamin B-12 and its biomarkers. CONCLUSIONS: Deficient and marginal serum vitamin B-12 concentrations are prevalent in Canadian pregnant women with the use of traditional cutoffs, despite supplement use. Given the growing interest among women to adhere to a vegetarian diet that may be lower in vitamin B-12, and vitamin B-12's importance in pregnancy, the functional ramifications of these observations need to be elucidated. This trial was registered at clinicaltrials.gov as NCT02244684.