Screening of Pharmaceutical Pollutants Along with Emerging Contaminants in the Sediments of the Periyar River, Located in Kerala (India) by Using High-Resolution Mass Spectrometry (UPLC-Q-ToF-MS).

In this study, ultra-performance liquid chromatography coupled with a quadruple time-of-flight mass spectrometry (UPLC-Q-ToF-MS) was employed to screen and identify pharmaceutical pollutants and emerging contaminants (ECs) in the Periyar River near Aster Medicity hospital, the dumping yard of Amrutha hospital, and the Vaduthala bridge regions in Kerala, India. The analysis was conducted in both positive and negative ionization modes using electrospray ionization (ESI). The QuEChERS method was employed for initial sediment sample preparation. Among the twenty-five identified ECs, four compounds were identified as pharmaceutical pollutants. This study have great significance as it represents the first comprehensive investigation of pharmaceutical pollutants in these hospital regions, highlighting the urgent need for further analysis and understanding of the situation. The presence of ECs poses an urgent need for attention due to the irreversible harm caused to the riverine ecosystem by the degradation of water quality resulting from industrial and domestic discharge.

Discovery of a Thermostable GH10 Xylanase with Broad Substrate Specificity from the Arctic Mid-Ocean Ridge Vent System.

A two-domain GH10 xylanase-encoding gene (amor_gh10a) was discovered from a metagenomic data set, generated after in situ incubation of a lignocellulosic substrate in hot sediments on the sea floor of the Arctic Mid-Ocean Ridge (AMOR). AMOR_GH10A comprises a signal peptide, a carbohydrate-binding module belonging to a previously uncharacterized family, and a catalytic glycosyl hydrolase (GH10) domain. The enzyme shares the highest sequence identity (42%) with a hypothetical protein from a Verrucomicrobia bacterium, and its GH10 domain shares low identity (24 to 28%) with functionally characterized xylanases. Purified AMOR_GH10A showed thermophilic and halophilic properties and was active toward various xylans. Uniquely, the enzyme showed high activity toward amorphous cellulose, glucomannan, and xyloglucan and was more active toward cellopentaose than toward xylopentaose. Binding assays showed that the N-terminal domain of this broad-specificity GH10 binds strongly to amorphous cellulose, as well as to microcrystalline cellulose, birchwood glucuronoxylan, barley ß-glucan, and konjac glucomannan, confirming its classification as a novel CBM (CBM85).IMPORTANCE Hot springs at the sea bottom harbor unique biodiversity and are a promising source of enzymes with interesting properties. We describe the functional characterization of a thermophilic and halophilic multidomain xylanase originating from the Arctic Mid-Ocean Ridge vent system, belonging to the well-studied family 10 of glycosyl hydrolases (GH10). This xylanase, AMOR_GH10A, has a surprisingly wide substrate range and is more active toward cellopentaose than toward xylopentaose. This substrate promiscuity is unique for the GH10 family and could prove useful in industrial applications. Emphasizing the versatility of AMOR_GH10A, its N-terminal domain binds to both xylans and glycans, while not showing significant sequence similarities to any known carbohydrate-binding module (CBM) in the CAZy database. Thus, this N-terminal domain lays the foundation for the new CBM85 family.

Dose response effects of cognitive-behavioral therapy in a school mental health program.

School mental health (SMH) programs have been shown to be effective in providing evidence-based interventions to underserved youth. However, limitations of SMH programs are that they can entail holiday breaks, typically do not operate through summer, and often require pulling students from class to receive therapy. These limitations suggest that treatment must be expeditious and potent. Although researchers have investigated dose response to treatment, no studies were located that addressed dose response to treatment in SMH programs. The present study addressed this gap by evaluating the dose response to SMH treatment in a sample of 133 adolescents. Adolescents were assessed at baseline, post-treatment, and at multiple time points throughout treatment. An average treatment response of a 26.81-point decrease in Youth Outcome Questionnaire (YOQ-30) score was found across 14 sessions of cognitive-behavioral therapy (CBT). Further, adolescents exhibited reliable change in YOQ-30 score within an average of 2.91 sessions. Finally, it was found that baseline scores on the Depression and Hyperactivity subscales of the Behavior Assessment System for Children, 2nd Edition, along with YOQ-30 score, predicted treatment response. These findings advance our understanding of dose response to CBT in SMH settings, and create opportunities to better inform effective treatment strategies in similar contexts.

Biochemical and Cellular Characterization and Inhibitor Discovery of Pseudomonas aeruginosa 15-Lipoxygenase.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause nosocomial and chronic infections in immunocompromised patients. P. aeruginosa secretes a lipoxygenase, LoxA, but the biological role of this enzyme is currently unknown. LoxA is poorly similar in sequence to both soybean LOX-1 (s15-LOX-1) and human 15-LOX-1 (37 and 39%, respectively) yet has kinetics comparably fast versus those of s15-LOX-1 (at pH 6.5, Kcat = 181 ± 6 s(-1) and Kcat/KM = 16 ± 2 µM(-1) s(-1)). LoxA is capable of efficiently catalyzing the peroxidation of a broad range of free fatty acid (FA) substrates (e.g., AA and LA) with high positional specificity, indicating a 15-LOX. Its mechanism includes hydrogen atom abstraction [a kinetic isotope effect (KIE) of >30], yet LoxA is a poor catalyst against phosphoester FAs, suggesting that LoxA is not involved in membrane decomposition. LoxA also does not react with 5- or 15-HETEs, indicating poor involvement in lipoxin production. A LOX high-throughput screen of the LOPAC library yielded a variety of low-micromolar inhibitors; however, none selectively targeted LoxA over the human LOX isozymes. With respect to cellular activity, the level of LoxA expression is increased when P. aeruginosa undergoes the transition to a biofilm mode of growth, but LoxA is not required for biofilm growth on abiotic surfaces. However, LoxA does appear to be required for biofilm growth in association with the host airway epithelium, suggesting a role for LoxA in mediating bacterium-host interactions during colonization.

A high-throughput mass spectrometric assay for discovery of human lipoxygenase inhibitors and allosteric effectors.

Lipoxygenases (LOXs) regulate inflammation through the production of a variety of molecules whose specific downstream effects are not entirely understood due to the complexity of the inflammation pathway. The generation of these biomolecules can potentially be inhibited and/or allosterically regulated by small synthetic molecules. The current work describes the first mass spectrometric high-throughput method for identifying small molecule LOX inhibitors and LOX allosteric effectors that change the substrate preference of human lipoxygenase enzymes. Using a volatile buffer and an acid-labile detergent, enzymatic products can be directly detected using high-performance liquid chromatography-mass spectrometry (HPLC-MS) without the need for organic extraction. The method also reduces the required enzyme concentration compared with traditional ultraviolet (UV) absorbance methods by approximately 30-fold, allowing accurate binding affinity measurements for inhibitors with nanomolar affinity. The procedure was validated using known LOX inhibitors and the allosteric effector 13(S)-hydroxy-9Z,11E-octadecadienoic acid (13-HODE).

Identification of high-risk Dukes B colorectal cancer by microRNA expression profiling: a preliminary study.

AIM: MicroRNAs (miRNAs) from tumour tissue and common gene mutations were studied to determine whether they predict the development of metastasis in patients with Dukes B colorectal cancer. METHOD: Patients who underwent curative resection for Dukes B colorectal cancer who subsequently developed distant metastatic disease at some stage in the following 5 years ('high-risk B') were compared with case-matched controls of Dukes A, Dukes B (no metastases, 'low-risk B') and Dukes C patients without any detectable metastasis at 5 years of follow-up. MiRNAs from tumour and adjacent normal tissue and common gene mutations (KRAS, BRAF, PIK3CA) in primary cancer tissue were analysed to identify prognostic tissue markers for the development of metastasis in patients with Dukes B colorectal cancer. RESULTS: Expression of miR-15b and miR-135b was significantly downregulated (P < 0.001) in 'high-risk B' tumours compared with Dukes A, 'low-risk B' and C without metastasis. No significant differences were noted for mutation status and the development of metastasis. CONCLUSION: The study suggests that the development of metastasis in Dukes B tumours may be predictable based on the miRNA expression of miR-15b and miR-135b. This requires further study on a much larger cohort.

Characterization and application of a disease-cell model for a neurodegenerative lysosomal disease.

Disease-cell models that recapitulate specific molecular phenotypes are essential for the investigation of molecular pathogenesis of neurodegenerative diseases including lysosomal storage diseases (LSDs) with predominant neurological manifestations. Herein we report the development and characterization of a cell model for a rapid neurodegenerative LSDs, globoid-cell leukodystrophy (GLD), mostly known as Krabbe disease. GLD is caused by the deficiency of ß-galactocerebrosidase (GALC), a lysosomal enzyme that hydrolyzes two glycosphingolipids, psychosine and galactosylceramide. Unfortunately, the available culture fibroblasts from GLD patients consist of a limited research tool as these cells fail to accumulate psychosine, the central pathogenic glycosphingolipid in this LSD that results in severe demyelination. Firstly, we obtained brain samples from the Twitcher (Twi) mice (GALC(twi/twi)), the natural mouse model with GALC deficiency. We immortalized the primary neuroglial cultured cells with SV40 large T antigen, generating the 145M-Twi and the 145C-Wt cell lines from the Twi and control mice, respectively. Both cell lines expressed specific oligodendrocyte markers including A2B5 and GalC. The 145M-Twi cells showed biochemical and cellular disturbances related to GLD neuropathogenesis including remarkable caspase-3 activation, release of cytochrome C into the cytosol and expansion of the lysosomal compartment. Under treatment with glycosphingolipids, 145M-Twi cells showed increased LC3B levels, a marker of autophagy. Using the LC-MS/MS method that we developed, the 145M-Twi cells showed significantly higher levels of psychosine. The 145M-Twi and 145C-Wt lines allowed the development of a robust throughput LC-MS/MS assay to measure cellular psychosine levels. In this throughput assay, l-cycloserine showed to significantly reduce the 145M-Twi cellular levels of psychosine. The established 145M-Twi cells are powerful research tools to investigate the neurologically relevant pathogenic pathways as well as to develop primary screening assays for the identification of therapeutic agents for GLD and potentially other glycosphingolipid disorders.

Peri-operative care of the elderly 2014: Association of Anaesthetists of Great Britain and Ireland.

Increasing numbers of elderly patients are undergoing an increasing variety of surgical procedures. There is an age-related decline in physiological reserve, which may be compounded by illness, cognitive decline, frailty and polypharmacy. Compared with younger surgical patients, the elderly are at relatively higher risk of mortality and morbidity after elective and (especially) emergency surgery. Multidisciplinary care improves outcomes for elderly surgical patients. Protocol-driven integrated pathways guide care effectively, but must be individualised to suit each patient. The AAGBI strongly supports an expanded role for senior geriatricians in coordinating peri-operative care for the elderly, with input from senior anaesthetists (consultants/associate specialists) and surgeons. The aims of peri-operative care are to treat elderly patients in a timely, dignified manner, and to optimise rehabilitation by avoiding postoperative complications. Effective peri-operative care improves the likelihood of very elderly surgical patients returning to their same pre-morbid place of residence, and maintains the continuity of their community care when in hospital. Postoperative delirium is common, but underdiagnosed, in elderly surgical patients, and delays rehabilitation. Multimodal intervention strategies are recommended for preventing postoperative delirium. Peri-operative pain is common, but underappreciated, in elderly surgical patients, particularly if they are cognitively impaired. Anaesthetists should administer opioid-sparing analgesia where possible, and follow published guidance on the management of pain in older people. Elderly patients should be assumed to have the mental capacity to make decisions about their treatment. Good communication is essential to this process. If they clearly lack that capacity, proxy information should be sought to determine what treatment, if any, is in the patient's best interests. Anaesthetists must not ration surgical or critical care on the basis of age, but must be involved in discussions about the utility of surgery and/or resuscitation. The evidence base informing peri-operative care for the elderly remains poor. Anaesthetists are strongly encouraged to become involved in national audit projects and outcomes research specifically involving elderly surgical patients.

Dax1 is required for testis determination.

The orphan nuclear receptor, Dax1, was originally proposed to act as an 'anti-testis' factor. We find, however, that Nr0b1 (also called Dax1 and Ahch, which encodes Dax1) is in fact required for testis differentiation.

Role of Ahch in gonadal development and gametogenesis.

Ahch (also known as Dax1) encodes a transcription factor that has been implicated in sex determination and gonadal differentiation. Mutations in human AHC cause X-linked, adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH). Duplication of the Xp21 dosage-sensitive sex reversal (DSS) region, which contains the Ahch locus, and transgenic overexpression of Ahch cause male-to-female sex reversal. Using Cre-mediated disruption of Ahch, we have generated a mouse model of AHC-HH that allows the function of Ahch to be examined in both males and females. Although Ahch has been postulated to function as an ovarian determination gene, the loss of Ahch function in females does not affect ovarian development or fertility. Ahch is instead essential for the maintenance of spermatogenesis. Lack of Ahch causes progressive degeneration of the testicular germinal epithelium independent of abnormalities in gonadotropin and testosterone production and results in male sterility. Ahch is thus not an ovarian determining gene, but rather has a critical role in spermatogenesis.

Addressing Structural Racism Using a Whole-Scale Planning Process in a Single Academic Center.

Purpose: The murder of George Floyd in 2020 prompted a national demand for cultural transformation to confront the systemic racism prevalent in the country. Academic medical centers were not exempt from this urgent call. This article evaluates the efficacy of a strategic process in fostering cultural transformation within an academic medical system. Methods: A whole-scale strategic planning process was implemented over 13 months, involving multiple working groups representing key stakeholders from each entity across the system, an anonymous survey, a communication plan, and a balanced scorecard to monitor progress. More than 5500 voices, 160 recommendations, 122 data gathering sessions, and town hall meetings contributed to the creation and implementation of vital action items and a strategic framework. The Diversity Engagement Survey (DES) was administered 18 months following the process launch. Results: Of the 45,554 employees, students, faculty, and trainees, 96.5% completed unconscious bias education within the fiscal year and 76% of action items, termed "Just Do Its," were completed. Mission, vision, values, and strategic priorities were crafted to serve as a framework for intermediate and long-term actions. The DES revealed improvement in the "respect" attribute of an inclusive culture, and 64% of respondents confirmed that action for cultural transformation is addressing racism both within and outside of the institution. Conclusion: Implementing a shared purpose, engaging multiple working groups representing key stakeholders, and empowerment of stakeholders to implement changes, in conjunction with the development of a strategic framework addressing structural racism, resulted in the completion of vital action items to initiate cultural change.

Teaching argument writing in math class: challenges and solutions to improve the performance of 4th and 5th graders with disabilities.

Incorporating argument writing as a learning activity has been found to increase students' mathematics performance. However, teachers report receiving little to no preservice or inservice preparation to use writing to support students' learning. This is especially concerning for special education teachers who provide highly specialized mathematics instruction (i.e., Tier 3) to students with mathematics disabilities (MLD). The purpose of this study was to examine the effectiveness of teachers providing content-focused open-ended questioning strategies, which included both argument writing and foundational fraction content, using Practice-Based Professional Development (PBPD) and Self-Regulated Strategy Development (SRSD) for implementing a writing-to-learn strategy called FACT-R2C2. We report the relative number of higher-order mathematical content questions that teachers asked during instruction, from among three different-level question types: Level 1: yes/no questions focused on the mathematics content; Level 2: one-word responses focused on the mathematics content; and Level 3: higher-order open-ended responses centered around four mathematical practices from the Common Core State Standards for Mathematics. Within a well-controlled single-case multiple-baseline design, seven special education teachers were randomly assigned to each PBPD + FACT-R2C2 intervention tier. Results indicated that: (1) teachers' relative use of Level 3 questions increased following the introduction of the FACT intervention; (2) this increase was apart from the professional development training that the teachers had initially received; and (3) students' writing quality improved to some extent with the increase in teachers' relative use of Level 3 questions. Implications and future directions are discussed.

Analytical approaches for the evaluation of data deficient simulated leachable compounds in ENDS products: a case study.

Leachable investigations are routinely undertaken across a range of sectors (e.g., pharmaceuticals, medical devices, etc.) to determine whether chemicals from a container closure system transfer into a product under normal conditions of use. For Electronic Nicotine Delivery Systems (ENDS) the container closure system includes all materials in contact with the e-liquid that is aerosolized and subsequently inhaled by the user. Currently, there is no guidance for conducting leachable studies for ENDS products, however, there are relevant guidance documents for orally inhaled drug products that can be applied to an ENDS container closure system. We present a case study of the analytical investigation of two leachable compounds identified in simulated leachable studies using aged JUULpods filled with unflavored e-liquid (PG/VG/nicotine/benzoic acid). Both compounds had limited toxicological information and were considered data deficient. A qualitative analysis of the aerosol collected from aged commercial JUULpods (Virginia Tobacco and Menthol), using a similar analytical method (LC-MS/MS) used in the simulated leachable studies, showed no trace or detectable levels of either leachable compound. Therefore, this qualitative analysis did not provide semi-quantitative values for the data-deficient leachable compounds necessary to support toxicological risk assessment. Further, no commercial authentic standards or reasonable synthetic route were available due to the molecular size and structural complexity of the compounds. Instead, method limits were established using an alternative approach to standard ICH guidelines. The experimentally determined method limit of quantitation, using spiked samples of simulated leachable e-liquid, provided conservative semi-quantitative values for each data deficient leachable compound in the aerosol that enabled a transfer efficiency from e-liquid to aerosol to be estimated. The transfer efficiency of each leachable compound was experimentally determined to be less than 2% based on the limit of quantitation, which then could be used to define a relevant exposure limit for the toxicological risk assessment. This work details a novel analytical approach for determining the transfer efficiency of data deficient leachable compounds from ENDS container closure systems into the ENDS aerosol to support toxicological health risk assessments.

Determination of chemical constituent yields in e-cigarette aerosol using partial and whole pod collections, a comparative analysis.

Literature reports the chemical constituent yields of electronic nicotine delivery systems (ENDS) aerosol collected using a range of aerosol collection strategies. The number of puffs to deplete an ENDS product varies widely, but collections often consist of data from the first 50-100 puffs. However, it is not clear whether these discrete puff blocks are representative of constituent yields over the life of a pod. We aimed to assess the effect of differing aerosol collection strategies on reported yields for select chemical constituents in the aerosol of closed pod-based ENDS products. Constituents analyzed were chosen to reflect important classes of compounds from the Final Premarket Tobacco Product Application Guidance. Yields were normalized to total device mass loss (DML). Collection strategies that consisted of partial pod collection were valid for determining yields of constituents whose DML normalized yields were consistent for the duration of pod life. These included primary aerosol constituents, such as propylene glycol, glycerol, and nicotine, and whole pod yields could be determined from initial puff blocks. However, changes were observed in the yields of some metals, some carbonyl compounds, and glycidol over pod life in a chemical constituent and product dependent manner. These results suggest that collection strategies consisting of initial puff block collections require validation per chemical constituent/product and are not appropriate for chemical constituents with variable yields over pod life. Whole pod collection increased sensitivity and accuracy in determining metal, carbonyl, and glycidol yields compared to puff block-based collection methodologies for all products tested.

Corrigendum: Analytical approaches for the evaluation of data deficient simulated leachable compounds in ENDS products: a case study.

[This corrects the article DOI: 10.3389/fchem.2023.1212744.].

ENU mutagenesis in mice identifies candidate genes for hypogonadism.

Genome-wide mutagenesis was performed in mice to identify candidate genes for male infertility, for which the predominant causes remain idiopathic. Mice were mutagenized using N-ethyl-N-nitrosourea (ENU), bred, and screened for phenotypes associated with the male urogenital system. Fifteen heritable lines were isolated and chromosomal loci were assigned using low-density genome-wide SNP arrays. Ten of the 15 lines were pursued further using higher-resolution SNP analysis to narrow the candidate gene regions. Exon sequencing of candidate genes identified mutations in mice with cystic kidneys (Bicc1), cryptorchidism (Rxfp2), restricted germ cell deficiency (Plk4), and severe germ cell deficiency (Prdm9). In two other lines with severe hypogonadism, candidate sequencing failed to identify mutations, suggesting defects in genes with previously undocumented roles in gonadal function. These genomic intervals were sequenced in their entirety and a candidate mutation was identified in SnrpE in one of the two lines. The line harboring the SnrpE variant retains substantial spermatogenesis despite small testis size, an unusual phenotype. In addition to the reproductive defects, heritable phenotypes were observed in mice with ataxia (Myo5a), tremors (Pmp22), growth retardation (unknown gene), and hydrocephalus (unknown gene). These results demonstrate that the ENU screen is an effective tool for identifying potential causes of male infertility.

p21-Activated kinase mediates rapid estradiol-negative feedback actions in the reproductive axis.

Nonclassical estrogen receptor alpha (ERalpha) signaling can mediate E(2) negative feedback actions in the reproductive axis; however, downstream pathways conveying these effects remain unclear. These studies tested the hypothesis that p21-activated kinase 1 (PAK1), a serine/threonine kinase rapidly activated by E(2) in nonneural cells, functions as a downstream node for E(2) signaling pathways in cells of the preoptic area, and it may thereby mediate E(2) negative feedback effects. Treatment of ovariectomized (OVX) rats with estradiol benzoate (EB) caused rapid and transient induction of phosphorylated PAK1 immunoreactivity in the medial preoptic nucleus (MPN) but not the arcuate nucleus. To determine whether rapid induction of PAK phosphorylation by E(2) is mediated by nonclassical [estrogen response element (ERE)-independent] ERalpha signaling, we used female ERalpha null (ERalpha(-/-)) mice possessing an ER knock-in mutation (E207A/G208A; AA), in which the mutant ERalpha is incapable of binding DNA and can signal only through membrane-initiated or ERE-independent genotropic pathways (ERalpha(-/AA) mice). After 1-h EB treatment, the number of pPAK1-immunoreactive cells in the MPN was increased in both wild-type (ERalpha(+/+)) and ERalpha(-/AA) mice but was unchanged in ERalpha(-/-) mice. Serum luteinizing hormone (LH) was likewise suppressed within 1 h after EB treatment in ERalpha(+/+) and ERalpha(-/AA) but not ERalpha(-/ -) mice. In OVX rats, 5-min intracerebroventricular infusion of a PAK inhibitor peptide but not control peptide blocked rapid EB suppression of LH secretion. Taken together, our findings implicate PAK1 activation subsequent to nonclassical ERalpha signaling as an important component of the negative feedback actions of E(2) in the brain.

CALM gatekeeper training is associated with increased confidence in utilizing means reduction approaches to suicide prevention among college resident assistants.

BACKGROUND: Most suicide prevention programs focus on increasing knowledge regarding the problem of suicide, yet many fail to include information on the science and application of means reduction approaches. In an attempt to address this gap in practice, the Counseling on Access to Lethal Means (CALM) program was developed to educate clinicians on the importance of means reduction interventions. METHODS: In the current study, a gatekeeper CALM training was delivered to 167 resident assistants. Confidence levels regarding suicide prevention and means reduction skills were assessed at baseline, post-training, and after a 6-week follow-up. RESULTS: Results were suggestive of medium to large training effects. Though there was a small decay of training effects at follow-up, the effects were durable when compared to baseline levels. CONCLUSION: Given these findings, future gatekeeper trainings should be provided more consistently to help sustain the effects and data on the implementation of CALM principles should be measured during follow-up assessments.