A mixed methods investigation of college student mental health during the first year of the COVID-19 pandemic.

OBJECTIVE: Researchers collaborated with undergraduate minority students to quantitatively and qualitatively investigate college students' mental health during the first year of the COVID-19 pandemic. PARTICIPANTS: Participants were two convenience samples of diverse college students surveyed in June (n = 128; M age = 21.7, SD = 1.7) and December (n = 242; M age = 20.3, SD = 1.7) of 2020. METHODS: This study administered items from the California Health Interview Survey and open-ended qualitative items via Qualtrics. RESULTS: Across both waves, students reported significant mental health challenges and psychological distress. Students surveyed in December were three to four times more likely to report depression and anxiety. Female and older students reported heightened odds of mental health challenges. Qualitative findings elaborated on contributing factors. CONCLUSION: During the pandemic, college students have experienced pronounced mental health challenges, potentially exacerbated by academic, professional, relational, and environmental stressors and uncertainty.

Similar social presence effects when reaching for real and digital objects.

Though digital images and real objects are represented differently at a neural level and can evoke different behaviours, little work has directly compared the magnitude of social effects on real and digitally represented stimuli. Object-directed reaches are modified in the near space of others, while image-directed reaches are not, but the exact role of the presence versus location of the other person is unknown (Dosso and Kingstone, 2018). The present work probed the unique contribution of social presence (a passive observer) in shaping object- and image-directed reaching behaviour. In a shape-matching game, movements were performed more slowly and less efficiently when participants were observed by the experimenter, regardless of whether participants handled real objects or digital images. Our finding that social presence affects real- and image-directed reaches similarly supports the continued use of computer-generated objects to approximate human behaviour towards real objects when social effects on object-directed actions are studied.

Investigation of biological factors influencing the placental mRNA profile in maternal plasma.

OBJECTIVE: Circulating placental-derived RNA is useful for noninvasive prenatal investigation. However, in addition to placental gene expression, there are limited investigations on other biological parameters that may affect the circulating placental RNA profile. In this study, we explored two of these potential parameters. METHODS: We first demonstrated the existence of such biological parameters by comparing the relative levels of a panel of placental-derived transcripts between the placentas and maternal plasma by digital PCRs. We then compared the post-delivery clearance of the transcripts by serial plasma samples collected from pregnant women after delivery. We also studied the placental in vivo localization of the transcripts by in situ hybridization. RESULTS: There was an imperfect correlation of the transcript levels between the placentas and maternal plasma, with placenta-specific 4 (PLAC4) mRNA showing the largest discrepancy. Although PLAC4 mRNA showed a similar clearance half-life with other transcripts, we observed a preferential localization of PLAC4 mRNA around the villous surface. We speculated that this phenomenon might play a role in favoring the release of PLAC4 mRNA molecules into maternal plasma. CONCLUSION: We revealed that in addition to expression levels in the placenta, other biological factors might interplay to determine the maternal plasma profile of placental-derived RNAs.

High resolution size analysis of fetal DNA in the urine of pregnant women by paired-end massively parallel sequencing.

BACKGROUND: Fetal DNA in maternal urine, if present, would be a valuable source of fetal genetic material for noninvasive prenatal diagnosis. However, the existence of fetal DNA in maternal urine has remained controversial. The issue is due to the lack of appropriate technology to robustly detect the potentially highly degraded fetal DNA in maternal urine. METHODOLOGY: We have used massively parallel paired-end sequencing to investigate cell-free DNA molecules in maternal urine. Catheterized urine samples were collected from seven pregnant women during the third trimester of pregnancies. We detected fetal DNA by identifying sequenced reads that contained fetal-specific alleles of the single nucleotide polymorphisms. The sizes of individual urinary DNA fragments were deduced from the alignment positions of the paired reads. We measured the fractional fetal DNA concentration as well as the size distributions of fetal and maternal DNA in maternal urine. PRINCIPAL FINDINGS: Cell-free fetal DNA was detected in five of the seven maternal urine samples, with the fractional fetal DNA concentrations ranged from 1.92% to 4.73%. Fetal DNA became undetectable in maternal urine after delivery. The total urinary cell-free DNA molecules were less intact when compared with plasma DNA. Urinary fetal DNA fragments were very short, and the most dominant fetal sequences were between 29 bp and 45 bp in length. CONCLUSIONS: With the use of massively parallel sequencing, we have confirmed the existence of transrenal fetal DNA in maternal urine, and have shown that urinary fetal DNA was heavily degraded.

Modification by antioxidant supplementation of changes in human lung function associated with air pollutant exposure: a systematic review.

BACKGROUND: Outdoor air pollution, given its demonstrated negative effects on the respiratory system, is a growing public health concern worldwide, particularly in urban cities. Human exposure to pollutants such as ozone, nitrogen oxides, combustion-related particulate matter and oxides of sulfur is responsible for significant cardiopulmonary morbidity and mortality in both adults and children. Several antioxidants have shown an ability to partially attenuate the negative physiological and functional impacts of air pollutants. This study systematically presents current data on the potential benefits of antioxidant supplementation on lung function outcomes associated with air pollutant exposures in intact humans. METHODS: Electronic databases (MEDLINE, EMBASE, BIOSIS Previews, Web of Sciences, Environmental Sciences & Pollution Management and TOXNET) were systematically searched for all studies published up to April 2009. Search terms relating to the concepts of respiratory tract diseases, respiratory function tests, air pollution, and antioxidants were used. Data was systematically abstracted from original articles that satisfied selection criteria for inclusion. For inclusion, the studies needed to have evaluated human subjects, given supplemental antioxidants, under conditions of known levels of air pollutants with measured lung function before and after antioxidant administration and/or air pollution exposure. Selected studies were summarized and conclusions presented. RESULTS: Eight studies investigated the role of antioxidant supplementation on measured lung function outcomes after subject exposure to air pollutants under controlled conditions; 5 of these studies concluded that pollutant-induced airway hyper-responsiveness and diminution in lung function measurements were attenuated by antioxidant supplementation. The remaining five studies took place under ambient (uncontrolled) exposures and unanimously concluded that antioxidant supplementations attenuate the negative effects of urban air pollution on lung function. CONCLUSIONS: The data evaluating modification of changes in lung function associated with air pollutant exposure by antioxidant supplementation, in intact humans, is limited. Of 13 studies dedicated to this concern, ten demonstrated an attenuation of pollution-associated decrements. There is growing evidence for the benefit of anti-oxidant supplementation in moderating the effects of air pollution on lung function, but more research on human participants is needed to inform this topic.

Synergy of total PLAC4 RNA concentration and measurement of the RNA single-nucleotide polymorphism allelic ratio for the noninvasive prenatal detection of trisomy 21.

BACKGROUND: Maternal plasma mRNA encoded by the PLAC4 gene (placenta-specific 4), which is transcribed from chromosome 21 in placental cells, is a potential marker for the noninvasive assessment of chromosome 21 dosage in the fetus. We evaluated the diagnostic sensitivities and specificities of 2 trisomy 21-screening approaches that use maternal plasma PLAC4 mRNA. METHODS: We studied maternal plasma samples from 153 pregnant women carrying euploid and trisomy 21 fetuses. For the samples in which the fetuses were heterozygous for the studied PLAC4 single-nucleotide polymorphism (SNP), we measured the ratio between 2 alleles of the SNP in maternal plasma PLAC4 mRNA (RNA-SNP) by mass spectrometric (MS) and digital PCR methods. For pregnancies involving fetuses homozygous for the SNP, we quantified the total PLAC4 mRNA concentration in maternal plasma by real-time PCR and digital PCR. RESULTS: For the RNA-SNP approach, we achieved a diagnostic sensitivity and specificity of 100% (95% CI, 40.2%-100%) and 89.7% (95% CI, 78.8%-96.1%), respectively, for both the MS and the digital PCR methods. For the mRNA-quantification approach, the areas under the ROC curves were 0.859 (95% CI, 0.741-0.903) and 0.833 (95% CI, 0.770-0.923) for plasma PLAC4 mRNA concentrations measured by the real-time PCR and the digital PCR methods, respectively. CONCLUSIONS: For prenatal screening of trisomy 21, the quantification of the total PLAC4 mRNA concentration can be used in a synergistic manner with the RNA-SNP allelic ratio approach to increase the population coverage of cases in which diagnostic information can be obtained.

Finding one's meaning: a test of the relation between quantifiers and integers in language development.

We explored children's early interpretation of numerals and linguistic number marking, in order to test the hypothesis (e.g., Carey (2004). Bootstrapping and the origin of concepts. Daedalus, 59-68) that children's initial distinction between one and other numerals (i.e., two, three, etc.) is bootstrapped from a prior distinction between singular and plural nouns. Previous studies have presented evidence that in languages without singular-plural morphology, like Japanese and Chinese, children acquire the meaning of the word one later than in singular-plural languages like English and Russian. In two experiments, we sought to corroborate this relation between grammatical number and integer acquisition within English. We found a significant correlation between children's comprehension of numerals and a large set of natural language quantifiers and determiners, even when controlling for effects due to age. However, we also found that 2-year-old children, who are just acquiring singular-plural morphology and the word one, fail to assign an exact interpretation to singular noun phrases (e.g., a banana), despite interpreting one as exact. For example, in a Truth-Value Judgment task, most children judged that a banana was consistent with a set of two objects, despite rejecting sets of two for the numeral one. Also, children who gave exactly one object for singular nouns did not have a better comprehension of numerals relative to children who did not give exactly one. Thus, we conclude that the correlation between quantifier comprehension and numeral comprehension in children of this age is not attributable to the singular-plural distinction facilitating the acquisition of the word one. We argue that quantifiers play a more general role in highlighting the semantic function of numerals, and that children distinguish between numerals and other quantifiers from the beginning, assigning exact interpretations only to numerals.

Noninvasive prenatal diagnosis of monogenic diseases by digital size selection and relative mutation dosage on DNA in maternal plasma.

Prenatal diagnosis of monogenic diseases, such as cystic fibrosis and beta-thalassemia, is currently offered as part of public health programs. However, current methods based on chorionic villus sampling and amniocentesis for obtaining fetal genetic material pose a risk to the fetus. Since the discovery of cell-free fetal DNA in maternal plasma, the noninvasive prenatal assessment of paternally inherited traits or mutations has been achieved. Due to the presence of background maternal DNA, which interferes with the analysis of fetal DNA in maternal plasma, noninvasive prenatal diagnosis of maternally inherited mutations has not been possible. Here we describe a digital relative mutation dosage (RMD) approach that determines if the dosages of the mutant and wild-type alleles of a disease-causing gene are balanced or unbalanced in maternal plasma. When applied to the testing of women heterozygous for the CD41/42 (-CTTT) and hemoglobin E mutations on HBB, digital RMD allows the fetal genotype to be deduced. The diagnostic performance of digital RMD is dependent on interplay between the fractional fetal DNA concentration and number of DNA molecules in maternal plasma. To achieve fetal genotype diagnosis at lower volumes of maternal plasma, fetal DNA enrichment is desired. We thus developed a digital nucleic acid size selection (NASS) strategy that effectively enriches the fetal DNA without additional plasma sampling or experimental time. We show that digital NASS can work in concert with digital RMD to increase the proportion of cases with classifiable fetal genotypes and to bring noninvasive prenatal diagnosis of monogenic diseases closer to reality.

Mass spectrometry-based detection of hemoglobin E mutation by allele-specific base extension reaction.

BACKGROUND: The specific detection of a minor population of mutant DNA molecules requires methods of high specificity and sensitivity. While the single-allele base extension reaction (SABER) was shown to be useful for the detection of certain beta-thalassemia mutations, we encountered problems with false positivity during development of SABER for the noninvasive prenatal diagnosis of the hemoglobin E (HbE) disease. Systematic optimization resulted in an alternative protocol, the allele-specific base extension reaction (ASBER). METHODS: An artificial model was established by mixing genomic DNA of HbE carriers and normal individuals. Effects of terminator concentration and annealing temperature on the nonspecificity of SABER were then studied. The use of a single relevant terminator and the other 3 types of dideoxynucleotide as competing terminators were also compared in the development of the ASBER protocol. Thirteen cases of HbE-susceptible pregnancies were tested to compare the SABER and the ASBER protocols. RESULTS: Decreasing the single relevant terminator concentration and increasing the annealing temperature in SABER were found to improve specificity. The use of the other 3 types of dideoxynucleotide as competing terminators was shown to offer better detection sensitivity than a single terminator in ASBER. Genotyping results were all correctly determined by ASBER, except one false-negative detection (sensitivity: 80%, specificity: 100%). CONCLUSIONS: An alternative mass spectrometry-based protocol for noninvasive prenatal diagnosis, ASBER, has been successfully developed to allow the detection of a minor DNA population with a point mutation.

Reduced plasma RNA integrity in nasopharyngeal carcinoma patients.

PURPOSE: Recent research has shown the feasibility of detecting cell-free RNA markers in human subjects. As elevated RNase activity has previously been described in the circulation of cancer patients, we hypothesized that cancer patients may have reduced plasma RNA integrity. In this study, we used nasopharyngeal carcinoma (NPC) as a model system to test this hypothesis. EXPERIMENTAL DESIGN: Plasma RNA integrity was determined using the ratio of the concentrations of transcript sequences corresponding to the 3' to those from the 5' end of a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Transcript concentrations were measured using real-time quantitative reverse transcription-PCR assays targeting the 5' and 3' regions. We analyzed the plasma RNA integrity in 49 untreated NPC patients and 53 healthy controls. We also assessed the plasma samples from 19 NPC patients before and after radiotherapy to further show the clinical potential of this marker. RESULTS: The 3' to 5' GAPDH ratio was significantly lower in the plasma of untreated NPC patients when compared with healthy controls (0.0252 versus 0.0485, P = 0.024). Statistical analysis showed that plasma GAPDH ratio was correlated with tumor stage but not with sex and age. Moreover, 14 of 19 NPC patients (74%) showed significant increase in the plasma GAPDH ratio following radiotherapy (P = 0.003). All of these patients were in clinical remission after treatment. CONCLUSIONS: Our findings suggest that NPC is associated with disturbances in the integrity of cell-free circulating RNA, raising the possibility that measurement of plasma RNA integrity may serve as a useful marker for the diagnosis and monitoring of malignant diseases.

Quantitative subtyping of hepatitis B virus reveals complex dynamics of YMDD motif mutants development during long-term lamivudine therapy.

BACKGROUND/AIMS: Treatment of chronic hepatitis B (CHB) with lamivudine (3TC) is limited by development of drug-resistant mutants at the YMDD motif. We aimed to validate the use of mass spectrometry to detect YMDD mutants and quantify viral subpopulations. METHODS: A total of 21 Chinese patients with severe acute exacerbation of CHB treated with 3TC were studied. Serial serum samples were tested for wild-type and YMDD mutants using matrix assisted laser desorption/ionization time-of-flight mass spectrometry. INNO-LiPA assay was performed for comparison. RESULTS: At a median follow-up of 192 weeks, 11 patients developed YMDD mutants (six had YIDD, four had YVDD and one had YV/IDD). Mass spectrometry was concordant with INNO-LiPA in all but one patient, in which INNO-LiPA detected coexistence of YIDD and YVDD but mass spectrometry and direct sequencing detected YVDD only. Mass spectrometry was able to reliably detect a minor hepatitis B virus (HBV) subtype at 5% or above. By serial quantitative measurement, several patterns of viral dynamics were observed. In some cases, YMDD mutants dominated the whole viral population. In other cases, the proportion of YMDD mutants fluctuated with time. When more than one mutant was present (that is, YIDD and YVDD), the different mutants might dominate during different time periods. CONCLUSIONS: Mass spectrometry is an accurate and cheap method for the detection of YMDD mutants, even in the presence of overwhelming wild-type HBV. We observed some intriguing mutant viral dynamics during 3TC treatment. Further studies are needed to clarify whether they have any clinical significance.

Quantitative analysis of cell-free Epstein-Barr virus DNA in plasma of patients with nonnasopharyngeal head and neck carcinomas.

PURPOSE: We investigated the detectability of EBV DNA in the plasma of patients with non-nasopharyngeal head and neck carcinomas (NNHNC). Previous studies have shown that EBV is present in the tumor tissue of some NNHNC. EXPERIMENTAL DESIGN: We recruited 101 patients with NNHNC and 48 healthy controls. Blood samples were taken from controls and patients before treatment. Tumor tissue samples were tested for the presence of EBV in the first 69 patients by in situ hybridization for small EBV-encoded RNA (EBER). Plasma EBV DNA was measured by real-time quantitative PCR in patients and controls. RESULTS: Squamous cell carcinoma (SCC) was the commonest histology (78 patients) followed by lymphoepithelial carcinoma (8 patients). EBER was detected in tumor cells in 7 of 69 patients tested. All of the EBER-positive tumors were lymphoepithelial carcinoma. Two controls (2 of 48; 4.2%) had detectable plasma EBV DNA. Plasma EBV DNA was detected in all of the patients with EBER-positive tumors, and in 23 of 94 (24.5%) patients with tumors of EBER-negative or unknown status. The proportion of plasma EBV DNA-positive cases in either group was significantly higher than that in the control group (P < 0.0027). Plasma EBV DNA concentrations in patients with EBER-positive tumors (median, 3827 copies/ml) were significantly higher than those in the controls (median, 0 copy/ml; P = 0.0001). Of patients with SCC, 21 (26.9%) had detectable plasma EBV DNA (median concentration, 34 copies/ml). Plasma EBV DNA concentrations in the whole group of patients with SCC (median, 0 copy/ml; interquartile range, 0-4 copies/ml) were also significantly higher than those in the controls (P = 0.001). CONCLUSIONS: Our data indicate that plasma EBV DNA reflects tumoral EBER status, and it may be of use as a tumor marker for EBER-positive NNHNC. The biological and clinical significance of low levels of circulating EBV DNA in the minority of patients with EBER-negative tumors remain to be elucidated.

Molecular characterization of circulating EBV DNA in the plasma of nasopharyngeal carcinoma and lymphoma patients.

Despite the increasing clinical applications of circulating EBV DNA analysis as a tumor marker, the molecular nature of these EBV DNA molecules remains unclear. We subjected plasma/serum samples of nasopharyngeal carcinoma and lymphoma patients to DNase digestion and ultracentrifugation and showed that circulating EBV DNA molecules are "naked" DNA fragments instead of being contained inside virions. We further showed that these EBV DNA fragments were relatively short, and 87% of them were shorter than 181 bp. These results provide fundamental information that may improve our understanding of the release of tumor-derived nucleic acids into the blood of cancer patients.

Quantitative analysis of pleural fluid cell-free DNA as a tool for the classification of pleural effusions.

BACKGROUND: Recently, much interest has been focused on the quantification of DNA in miscellaneous body fluids. In this study, the application is extended to classifying pleural effusions by measuring cell-free DNA in pleural fluid. METHODS: We recruited 50 consecutive patients with pleural effusions with informed consent. Pleural fluids were centrifuged at 13000 g, with supernatants aliquoted for extraction and analysis of beta-globin DNA sequence by quantitative real-time PCR. Serum and pleural fluid biochemistries were performed to classify pleural effusions using the modified criteria of Light et al. (Ann Intern Med 1972;77:507-13). The ROC curve was plotted to determine the cutoff DNA concentration for classifying pleural fluids as transudates or exudates. Indicators of diagnostic accuracy were calculated for both pleural fluid DNA and modified criteria of Light et al., using the discharge, microbiologic, and histologic diagnoses as the reference standard. RESULTS: The area under the ROC curve was 0.95 [95% confidence interval (CI), 0.84-0.99]. At 509 genome-equivalents/mL, pleural fluid DNA alone correctly classified 46 of 50 pleural effusions with 91% sensitivity (95% CI, 76-98%), 88% specificity (95% CI, 64-98%), and positive and negative likelihood ratios of 7.7 (95% CI, 3.1-19.5) and 0.10 (95% CI, 0.04-0.27), respectively. With the modified criteria of Light et al., 43 of 50 pleural effusions were correctly classified with 97% sensitivity (95% CI, 91-100%) and 67% specificity (95% CI, 45-89%). There were significant correlations between cell-free DNA and both lactate dehydrogenase and total protein in pleural fluid, suggesting their common origin. CONCLUSIONS: Pleural fluid DNA concentrations are markedly increased in exudative effusions, making it a potential new tool to evaluate the etiologic causes of pleural effusions.

Prenatal exclusion of beta thalassaemia major by examination of maternal plasma.

The discovery of the presence of fetal DNA in maternal plasma has provided a new approach for non-invasive prenatal diagnosis. At present, the prenatal diagnosis of beta thalassaemia relies on invasive methods. We designed allele-specific primers and a fluorescent probe for detection of the codon 41/42 (-CTTT) mutation in the beta globin gene from maternal plasma by real-time PCR. The specificity and sensitivity of the allele-specific assay was confirmed by subjecting plasma, buffy coat, and amniotic fluid samples from 100 pregnancies to screening for the mutation. Subsequently, the assay was applied to the prenatal testing of eight fetuses at risk of beta thalassaemia major, the aim being to exclude fetal inheritance of paternally transmitted codon 41/42 mutation. The fetal genotype was completely concordant with conventional analysis and beta thalassaemia major was excluded in two of the pregnancies non-invasively.

Differential DNA methylation between fetus and mother as a strategy for detecting fetal DNA in maternal plasma.

BACKGROUND: Fetal DNA has been detected in maternal plasma by the use of genetic differences between mother and fetus. We explore the possibility of using epigenetic markers for the specific detection of fetal DNA in maternal plasma. METHODS: A differentially methylated region in the human IGF2-H19 locus and a single-nucleotide polymorphism in this region were chosen for the study. The methylation status in this region is maintained in such a way that the paternal allele is methylated and the maternal allele is unmethylated. The single-nucleotide polymorphism was typed by direct sequencing of PCR products. The methylation status of this region was ascertained by bisulfite conversion and methylation-specific PCR. Differentially methylated fetal alleles were detected in maternal plasma by direct sequencing and a primer-extension assay. RESULTS: Women in the second (n = 21; 17-21 weeks) and third (n = 18; 37-42 weeks) trimesters of pregnancy were recruited. Among these 39 volunteers, the 16 who were heterozygous for the single-nucleotide polymorphism were chosen for further analysis. In 11 of these 16 cases, paternally inherited methylated fetal alleles were different from the methylated alleles of the respective mothers. Using direct sequencing, we detected paternally inherited methylated fetal DNA in 6 of 11 (55%) cases. In 8 of the 16 heterozygous cases, the fetuses possessed an unmethylated maternally inherited allele that was different from the unmethylated allele of the mother. Using a primer-extension assay, we detected fetal-derived maternally inherited alleles in maternal plasma of four of eight (50%) cases. CONCLUSIONS: These results represent the first use of fetal epigenetic markers in noninvasive prenatal analysis. These data may also have implications for the investigation of other types of chimerism.