BACKGROUND AND OBJECTIVE

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the causative agent of COVID-19 has significantly challenged the public health landscape in late 2019. After almost 3 years of the first ever SARS-CoV-2 case, the World Health Organization (WHO) declared the end of this global health emergency in May 2023. Although, despite the subsequent drop of COVID-19 cases, the SARS-CoV-2 infection still exhibited multiple waves of infection, primarily attributed to the appearance of new variants. Five of these variants have been classified as Variants of Concern (VOC): Alpha, Beta, Gamma, Delta, and the most recent, Omicron. Therefore, the development of methods for the timely and accurate detection of viral variants remains fundamental, ensuring an ongoing and effective response to the disease. This study aims to evaluate the feasibility of the application of an in-house approach in genomic surveillance for the detection of SARS-CoV-2 variants using in silico designed primers.

The primers used for the study were particularly designed based on conserved regions of certain genes in the virus, targeting distinct mutations found in known variants of SARS-CoV-2. Viral RNA extracts from nasopharyngeal samples (n=14) were subjected to quantitative and qualitative tests (Nanodrop and AGE). Selected samples were then analyzed by RT-PCR and amplicons were submitted for sequencing. Sequence alignment analysis was carried out to identify the prevailing COVID-19 variant present in the sample population.

The study findings demonstrated that the in-house method was able to successfully amplify conserved sequences (spike, envelope, membrane, ORF1ab) and enabled identification of the circulating SARS-CoV-2 variant among the samples. Majority of the samples were identified as Omicron variant. Three out of four designed primers effectively bound into the conserved sequence of target genes present in the sample, revealing the specific SARSCoV-2 variant. The detected mutations characterized for Omicron found in the identified lineages included K417N, S477N, and P681H which were also identified as mutations of interest. Furthermore, identification of the B.1.448 lineage which was not classified in any known variant also provided the potential of the developed in-house method in detecting unknown variants of COVID-19.

Among the five VOCs, Omicron is the most prevalent and dominant variant. The in-house direct PCR product sequencing surveillance (DPPSS) method provided an alternative platform for SAR-CoV-2 variant analysis which is accessible and affordable than the conventional diagnostic surveillance methods and the whole genome sequencing. Further evaluation and improvements on the oligonucleotide primers may offer significant contribution to the development of a specific and direct PCRbased detection of new emerging COVID-19 variants.

BACKGROUND AND OBJECTIVE

Endocrine Disrupting Chemicals (EDCs) are ubiquitously found as low-level contaminants and pose serious threat to women’s health. EDCs may result in various reproductive disorders, fetal birth and developmental abnormalities, and endocrine and metabolic disorders. EDCs can be detected in body fluids of exposed individuals including blood and urine. This study aimed to detect four EDCs — Methyl Paraben (MP), 2,4-Dichlorophenoxyacetic acid (2,4-D), Monobutyl Phthalate (MBP), and Bisphenol A (BPA) in urine samples of women using Ultra-Performance Liquid Chromatography – Quadrupole Time-of-Flight (UPLC-QTOF) mass spectrometry.

Sequential steps of enzymatic deconjugation, liquid-liquid extraction, solid phase extraction, and liquid chromatography separation and mass spectrometry detection were optimized in urine samples. The method was used to analyze 70 urine samples from women of reproductive age.

The sample preparation method showed a recovery ranging from 86.6% (MBP) to 100 % (2,4-D). The method demonstrated limits of quantitation ranging from 1.52 ng/m(MP) to 6.46 ng/mL(2,4D). Intra-day precisions expressed as relative standard deviation were all below 15% while accuracy was shown to range from 67.10% (2,4-D) to 102.39% (MBP). MP was detected in nine samples (12.86%) with a geometric mean value of 10.15 ng/ml (range: 3.62-52.39 ng/ml). MBP was detected in 68 samples (97.14%) with a geometric mean value of 97.62 ng/ml (range: 15.32-698.18 ng/ml). BPA was detected only once (9.58 ng/ml) while 2, 4-D was not detected in all samples.

A UPLC-QTOF mass spectrometry method to detect four EDCs at parts per billion level (ng/ml) was adapted and applied for analysis of urine samples. This method can find applicability in routine testing of clinical specimens as well as surveillance and other epidemiological studies.

BACKGROUND AND OBJECTIVES

Cell lines serve as invaluable tools in studying lung cancer biology and developing new therapies to combat the disease. However, commercially available cell lines are typically of Caucasian origin and may be less representative of the local genetic background. To address this, our lab previously immortalized cells from pleural fluid of a Filipino non-small cell lung cancer (NSCLC) patient via CDK4 transduction. Copy number variations (CNVs) are a type of genetic variation which may affect physiology and disease by disrupting gene function or altering gene expression, and in cancer, these may be associated with patient outcomes. CNV profiling can be valuable for understanding the biology of our immortalized cells and identifying genes that could serve as potential targets for diagnostic, prognostic, and therapeutic interventions. This study aimed to characterize previously immortalized NSCLC-derived cells, GL01, in comparison with an established lung adenocarcinoma (LUAD) cell line, A549, through whole-genome microarray-based copy number profiling.

DNA was extracted from GL01 and A549 cells using a commercially-available silica-based DNA extraction kit. DNA extracts were quantified and normalized for microarray analysis. Whole-genome copy number profiling was done using the OncoScan CNV Plus Assay following the manufacturer’s protocols, and data was analyzed using the Chromosome Analysis Suite software. Functional analysis of genes identified to be involved in copy number aberrations was done using the PANTHER Classification System.

Copy number aberrations span 1,592,737,105 bp in GL01 and 1,715,708,552 bp in A549, with a high degree of concordance between the two. Large-scale and focal copy number aberrations previously identified to be recurrent in various LUAD cohorts were present in both GL01 and A549. Focal copy number aberrations associated with previously described lung cancer-related genes involve the PDE4D gene in GL01 and the SKIL and CDKN2A/CDKN2B genes in both GL01 and A549. PANTHER Pathway analysis of genes positively correlated with mRNA expression showed that the ubiquitin proteasome pathway was significantly overrepresented in both GL01 (FDR p = 0.000074) and A549 (FDR p = 0.000075), with 20 genes involved. Additionally, the KRAS:p.G12C/S:c.34G>T/A somatic mutation variant was detected in both GL01 and A549.

This study provides a method for identifying potentially clinically-relevant genes associated with a sample’s copy number aberrations and the pathways they represent, providing personalized mechanistic, prognostic, and therapeutic insights into the cancer biology of our cells.

Background and Objective: Convalescent plasma therapy (CPT) may reduce the risk of disease progression among patients with COVID-19. This study was undertaken to evaluate the efficacy and safety of CPT in preventing ICU admission among hospitalized COVID-19 patients. Methods: In this open-label randomized controlled trial, we randomly assigned hospitalized adult patients with COVID-19 in a 1:1 ratio to receive convalescent plasma as an adjunct to standard of care or standard of care alone. The primary endpoint was ICU admission within first 28 days of enrolment. Primary safety endpoints include rapid deterioration of respiratory or clinical status within four hours of convalescent plasma transfusion and cumulative incidence of serious adverse events during the study period including transfusion-related acute lung injury (TRALI), transfusion-associated circulatory overload (TACO), severe allergic reactions, and transfusion-related infections. Results: A total of 22 patients were assigned to receive convalescent plasma as an adjunct to standard of care and 22 to receive standard of care alone. The median time from onset of COVID-19 symptoms to study enrolment was eight days (IQR, 4 to 10). Two patients (9.1%) in the CPT group and one patient (4.5%) in the control group were admitted to the ICU. The primary outcome measure, ICU admission, was not different between the two groups (q-value >0.9). No patient who received convalescent plasma had rapid deterioration of respiratory/clinical status within four hours of transfusion and none developed TRALI, TACO, anaphylaxis, severe allergic reactions, or transfusion-related infections. There was also no significant difference in the secondary outcomes of 28-day mortality (two patients in the CPT group and none in the control group, q-value >0.90), dialysis-free days, vasopressor-free days, and ICU-free days. Conclusions Among hospitalized COVID-19 patients, no significant differences were observed in the need for ICU admission between patients given CPT as adjunct to standard of care and those who received standard of care alone. Interpretation is limited by early termination of the trial which may have been underpowered to detect a clinically important difference.
COVID-19 ; COVID-19 Serotherapy

OBJECTIVES

To validate a method in detecting SARS-CoV-2 via RT-qPCR in pregnant and non-pregnant samples other than nasopharyngeal swabs and/or oropharyngeal swabs such as cervical, rectal, amniotic fluid, placental, umbilical cord blood, and breastmilk.

We performed a validation experiment using MGI easy extraction kits and BGI PCR kits on non-conventional specimens, including cervical, rectal, amniotic fluid, placental, umbilical cord blood, and breastmilk to detect and confirm the presence of SARS-CoV-2. In addition, we tested the validated method on 572 purposively sampled field-collected non-conventional specimens from a cohort of 109 unvaccinated pregnant and 47 unvaccinated non-pregnant women to assess which candidate non-conventional maternal- and fetal-associated specimens may contribute to maternal-fetal viral vertical transmission.

Positive detection of SARS-CoV-2 viral RNA in non-conventional specimens was demonstrated and verified. Of the 572 non-conventional samples tested, 1.8% (10/572) were positively validated by RT-qPCR for SARS-CoV-2 in the maternal-associated specimens particularly the rectal (5), placental (1), and cervical (4) swabs among six pregnant and four non-pregnant individuals. In contrast, no SARS-CoV-2 viral RNA was detected in fetal-associated specimens.

The results of the validation study may serve as an additional diagnostic screening layer to support maternal-child care. Furthermore, viral detection in these non-conventional maternal specimens may also be utilized to provide guidance in the clinical management of neonates, and pregnant women during delivery.

BACKGROUND: Polycystic ovarian syndrome (PCOS) is a prevalent condition affecting women of reproductive age, characterized by metabolic, endocrine, and reproductive disturbances including insulin resistance, abnormal uterine bleeding, infertility, and hyperandrogenism, and is associated with diabetes and cardiovascular disease. The etiology of PCOS is unclear and exposure to endocrine-disrupting chemicals (EDCs) present in everyday products may play a role by disrupting hormonal pathways. OBJECTIVES: To determine the association between exposure to EDC-containing products, sociodemographic factors, and PCOS diagnosis among nonpregnant reproductive-aged women. MATERIALS AND METHODS: A survey assessed the frequency of EDC exposure in homes and workplaces. The Rotterdam Criteria were used for the diagnosis of PCO features with clinical history and ultrasound. The association between PCOS and EDC exposure was determined using Chi-square and logistic regression analysis. RESULTS: The study identified significant sociodemographic factors associated with PCOS (P < 0.001) including age, civil status, and household income. Increased likelihood of PCOS was linked to frequent use of scented candles (odds ratio [OR] = 2.07), cleaning sprays (OR = 2.28), and floor polish (OR = 2.07), exposure to new upholstered furniture (OR = 4.00), thermal receipts (OR = 2.16), and consumption of microwaved and processed foods (OR = 2.60), as well as water sourced from wells (OR = 7.69). Additional associations were found with access to public markets (OR = 0.26) and the use of paper food wrappers (OR = 1.72). CONCLUSION These findings suggest that frequent exposure to EDC-containing products and certain sociodemographic factors may contribute to the development of PCOS among women of reproductive age. Results underscore the importance of reducing exposure to EDCs to prevent or mitigate the development of PCOS and other reproductive consequences.
Female

Background and Objective: Endocrine Disrupting Chemicals (EDCs) are ubiquitously found as low-level contaminants and pose serious threat to women’s health. EDCs may result in various reproductive disorders, fetal birth and developmental abnormalities, and endocrine and metabolic disorders. EDCs can be detected in body fluids of exposed individuals including blood and urine. This study aimed to detect four EDCs — Methyl Paraben (MP), 2,4-Dichlorophenoxyacetic acid (2,4-D), Monobutyl Phthalate (MBP), and Bisphenol A (BPA) in urine samples of women using Ultra-Performance Liquid Chromatography – Quadrupole Time-of-Flight (UPLC-QTOF) mass spectrometry. Methods: Sequential steps of enzymatic deconjugation, liquid-liquid extraction, solid phase extraction, and liquid chromatography separation and mass spectrometry detection were optimized in urine samples. The method was used to analyze 70 urine samples from women of reproductive age. Results: The sample preparation method showed a recovery ranging from 86.6% (MBP) to 100 % (2,4-D). The method demonstrated limits of quantitation ranging from 1.52 ng/m(MP) to 6.46 ng/mL(2,4D). Intra-day precisions expressed as relative standard deviation were all below 15% while accuracy was shown to range from 67.10% (2,4-D) to 102.39% (MBP). MP was detected in nine samples (12.86%) with a geometric mean value of 10.15 ng/ml (range: 3.62-52.39 ng/ml). MBP was detected in 68 samples (97.14%) with a geometric mean value of 97.62 ng/ml (range: 15.32-698.18 ng/ml). BPA was detected only once (9.58 ng/ml) while 2, 4-D was not detected in all samples. Conclusion A UPLC-QTOF mass spectrometry method to detect four EDCs at parts per billion level (ng/ml) was adapted and applied for analysis of urine samples. This method can find applicability in routine testing of clinical specimens as well as surveillance and other epidemiological studies.
Endocrine Disruptors

Background and Objective: Retinoblastoma is one of the most common intraocular cancers among children usually caused by the loss of retinoblastoma protein function. Despite being a highly heritable disease, conventional diagnostic and prognostic methods depend on clinical examination, with limited consideration of cancer genetics in the standard of care. CD133, KRT19, and MUC1 are commonly explored genes for their utility in liquid biopsies of cancer including lung adenocarcinoma. To date, there are few extensive molecular studies on retinoblastoma in Filipino patients. To this end, the study aimed to describe the copy number of CD133, KRT19, and MUC1 in retinoblastoma samples from a Filipino patient and quantitate the respective expression level of these genes. Methods: Hematoxylin & Eosin (H&E) staining was utilized to characterize the retinoblastoma tissue while fluorescence in situ hybridization (FISH) using probes specific to CD133, KRT19, and MUC1 was performed to determine the copy number of genes in retinoblastoma samples from a Filipino patient (n = 1). The gene expression of CD133, MUC1, and KRT19 was quantitated using RT-qPCR. Results: The H&E staining in the retinoblastoma tissue shows poorly differentiated cells with prominent basophilic nuclei. CD133 was approximately 1.5-fold overexpressed in the retinoblastoma tissue with respect to the normal tissue, while MUC1 and KRT19 are only slightly expressed. Multiple intense signals of each probe were localized in the same nuclear areas throughout the retinoblastoma tissue, with high background noise. Conclusion These findings suggest that CD133 is a potential biomarker for the staging and diagnosis of retinoblastoma in Filipino cancer patients. However, further optimization of the hybridization procedures is recommended.
Retinoblastoma ; Biomarkers ; In Situ Hybridization

Background and Objectives: Cell lines serve as invaluable tools in studying lung cancer biology and developing new therapies to combat the disease. However, commercially available cell lines are typically of Caucasian origin and may be less representative of the local genetic background. To address this, our lab previously immortalized cells from pleural fluid of a Filipino non-small cell lung cancer (NSCLC) patient via CDK4 transduction. Copy number variations (CNVs) are a type of genetic variation which may affect physiology and disease by disrupting gene function or altering gene expression, and in cancer, these may be associated with patient outcomes. CNV profiling can be valuable for understanding the biology of our immortalized cells and identifying genes that could serve as potential targets for diagnostic, prognostic, and therapeutic interventions. This study aimed to characterize previously immortalized NSCLC-derived cells, GL01, in comparison with an established lung adenocarcinoma (LUAD) cell line, A549, through whole-genome microarray-based copy number profiling. Methods: DNA was extracted from GL01 and A549 cells using a commercially-available silica-based DNA extraction kit. DNA extracts were quantified and normalized for microarray analysis. Whole-genome copy number profiling was done using the OncoScan CNV Plus Assay following the manufacturer’s protocols, and data was analyzed using the Chromosome Analysis Suite software. Functional analysis of genes identified to be involved in copy number aberrations was done using the PANTHER Classification System. Results: Copy number aberrations span 1,592,737,105 bp in GL01 and 1,715,708,552 bp in A549, with a high degree of concordance between the two. Largescale and focal copy number aberrations previously identified to be recurrent in various LUAD cohorts were present in both GL01 and A549. Focal copy number aberrations associated with previously described lung cancer-related genes involve the PDE4D gene in GL01 and the SKIL and CDKN2A/CDKN2B genes in both GL01 and A549. PANTHER Pathway analysis of genes positively correlated with mRNA expression showed that the ubiquitin proteasome pathway was significantly overrepresented in both GL01 (FDR p = 0.000074) and A549 (FDR p = 0.000075), with 20 genes involved. Additionally, the KRAS:p.G12C/S:c.34G>T/A somatic mutation variant was detected in both GL01 and A549. Conclusion This study provides a method for identifying potentially clinically-relevant genes associated with a sample’s copy number aberrations and the pathways they represent, providing personalized mechanistic, prognostic, and therapeutic insights into the cancer biology of our cells.
carcinoma, non-small cell lung ; adenocarcinoma of lung

Context: Exposure to environmental pollutants (EP) and Endocrine-disrupting chemicals (EDCs) is associated with several general negative health effects which compromise women's reproductive health, maternal, and neonatal outcomes. Unfortunately, many nonpregnant and pregnant women are unaware of their active exposure to these potentially slow-acting toxic substances, EPs, and EDCs. At any stage of life, and in the long-term minute exposures, there is no established safe level of exposure to these substances. Due to the potentially harmful effects on women in general, and to the pregnant and her unborn child in particular, it is important to establish the prevalence of their exposure. Aims: The aim of this study was to determine the magnitude (prevalence) of exposure among nonpregnant and pregnant women aged 18–49 years to common EPs/EDCs such as bisphenol A, pesticides, phthalates, and perfluorinated compounds among others. Settings and Design : This study was conducted at the Philippine General Hospital, University of the Philippines Manila. Subjects and Methods : The study participants were nonpregnant and pregnant women, with low-risk singleton pregnancy, and had a prenatal checkup and eventual delivery at the Philippine General Hospital. After consent, women were asked to answer a survey focused on their sociodemographics and frequency of exposure to EP- and EDC-containing items. Satatistical Analysis Used: Descriptive statistics were used to summarize the demographic and clinical characteristics of the patients. Null hypotheses were rejected at 0.05 α-level of significance. The computer software STATA 13.1 was used for data analysis. Results: One hundred and fifty-nine survey responses by women were analyzed. Possible EDC-containing household items (carpets, linoleum, upholstered, and stain-resistant furniture) are more prevalent in the homes of pregnant women compared to nonpregnant women. Pregnant women are also exposed to wallpapers compared to nonpregnant individuals. Nonpregnant women were 1.5 times more exposed to lotions. Unexposure to hand sanitizers is 1.3 times higher among pregnant individuals. Conclusions There are differences in the prevalence of exposure to household EDC-containing items between pregnant and nonpregnant women, with pregnant women having a higher prevalence of exposure.
Endocrine-disrupting chemicals ; environmental pollutants ; pregnancy