Awareness and Periodontal Health Practices of Fixed Orthodontic Appliance Patients: A Questionnaire-Based Survey.

A well-aligned dentition is more conducive to periodontal health, which is necessary for successful orthodontic therapy. Maintaining good dental hygiene is crucial for effective treatment, and patient cooperation, education, motivation, and attitude are all important components. Orthodontists must routinely check in with their patients to see whether they are maintaining their oral hygiene and if they are using any additional assistance. Negligence on the part of the patient may be the cause of poor treatment outcomes. This study aims to show how patient education can affect treatment outcomes and the development of a functional, aesthetically pleasing, and healthy dentition. Aim This study aims to investigate awareness, attitude, and periodontal health knowledge among orthodontic patients. Objectives The study explores the level of awareness and knowledge regarding periodontal health among orthodontic patients, examining its correlation with factors such as age, attitude, and duration of orthodontic treatment. Additionally, it aims to gauge the extent of education received by orthodontic patients regarding proper oral hygiene practices and the potential repercussions of neglecting them. Methods A questionnaire-based study with a cross-sectional design was performed in Jeddah, Saudi Arabia. A total of 428 participants were randomly selected from several private orthodontic clinics and hospitals. The participants in the study were individuals currently undergoing treatment with fixed orthodontic appliances. Information was gathered using a self-administered questionnaire. Results The patients undergoing orthodontic treatment have a moderate understanding of periodontal disease about dental plaque. The level of awareness of periodontal health was 41%, and the level of knowledge about periodontal health was 51%. When it came to periodontal health, adult orthodontic patients had a positive attitude toward fixed orthodontic treatment (mean score = 0.75). Subjects' attitudes regarding fixed orthodontic treatment were significantly impacted by the patient's consistency with his/her dental checkups (p value = 0.02). Conclusion The patients' periodontal health awareness was moderate, while their periodontal health knowledge was fair. Orthodontic patient's awareness levels, as well as knowledge levels, showed significant differences in age and duration but not sex. Results showed no significant difference among the attitude levels of orthodontic patients and age, sex, and duration.

Are we providing complete drug information to its users? Status of information adequacy of package insert in India.

Background: Package inserts (PIs) serve detailed information on drug products to the users and primary care physicians, so information should be accurate, reliable, and as per the regulatory guidelines. The study aims to analyze the information adequacy of the PIs available in the Indian market as per Drug and Cosmetic Rule 1945 and US Food and Drug Administration criteria. Materials and Methods: A cross-sectional study was conducted on PIs collected from accessible pharmacy stores. Information provided was recorded as per criteria, and total information adequacy score (IAS) and information deficiency (IDS) score were calculated. The association of factors like single-drug/FDCs, a company of origin Indian/multinational, and route of administration (ROA) with IDS was statistically analyzed. Results: Of 120 PIs, 60%, 86.66%, and 73% were single-drug, prescription-drug, and drugs by Indian manufacturers, respectively. Most PIs provided generic names, ROA, and indications for use. 85%, 12%, 29.16%, and 3.33% provided information on PIs on the ability to drive, drug-food interactions, drug-drug interactions, and addiction potential, respectively. Lacking area was information on use in pediatrics-geriatrics (30%), excipients (28.3%), preclinical (15.83%), post-surveillance data (18.33%), and approval date (2.5%). There was a statistically significant difference between pharmaceutical score (3.22 vs 4.12), therapeutic score (11.5 vs 13.18), and total IAS (14.78 ± 3.39 vs 17.31 ± 2.33) of Indian and multinational companies. IDS was statistically significantly different in both pharmaceutical and therapeutic categories for single-drug vs FDCs (P = 0.00001), OTC vs prescription drugs (P < 0.05), and Indian vs multinational companies' PIs (P = 0.00001). Conclusion: Numerous facets of information are lacking in PIs, and they do not impart whole information, especially of Indian origin, as per objective IDS.

Drivers of Antibiotic Resistance Gene Abundance in an Urban River.

In this study, we sought to profile the abundances and drivers of antibiotic resistance genes in an urban river impacted by combined sewage overflow (CSO) events. Water samples were collected weekly during the summer for two years; then, quantitative PCR was applied to determine the abundance of resistance genes associated with tetracycline, quinolones, and ß-lactam antibiotics. In addition to sampling a CSO-impacted site near the city center, we also sampled a less urban site ~12 km upstream with no proximal sewage inputs. The tetracycline genes tetO and tetW were rarely found upstream, but were common at the CSO-impacted site, suggesting that the primary source was untreated sewage. In contrast, ampC was detected in all samples indicating a more consistent and diffuse source. The two other genes, qnrA and blaTEM, were present in only 40-50% of samples and showed more nuanced spatiotemporal patterns consistent with upstream agricultural inputs. The results of this study highlight the complex sources of ARGs in urban riverine ecosystems, and that interdisciplinary collaborations across diverse groups of stakeholders are necessary to combat the emerging threat of antibiotic resistance through anthropogenic pollution.

Metformin: A Review of Potential Mechanism and Therapeutic Utility Beyond Diabetes.

Metformin has been designated as one of the most crucial first-line therapeutic agents in the management of type 2 diabetes mellitus. Primarily being an antihyperglycemic agent, metformin also has a plethora of pleiotropic effects on various systems and processes. It acts majorly by activating AMPK (Adenosine Monophosphate-Activated Protein Kinase) in the cells and reducing glucose output from the liver. It also decreases advanced glycation end products and reactive oxygen species production in the endothelium apart from regulating the glucose and lipid metabolism in the cardiomyocytes, hence minimizing the cardiovascular risks. Its anticancer, antiproliferative and apoptosis-inducing effects on malignant cells might prove instrumental in the malignancy of organs like the breast, kidney, brain, ovary, lung, and endometrium. Preclinical studies have also shown some evidence of metformin's neuroprotective role in Parkinson's disease, Alzheimer's disease, multiple sclerosis and Huntington's disease. Metformin exerts its pleiotropic effects through varied pathways of intracellular signalling and exact mechanism in the majority of them remains yet to be clearly defined. This article has extensively reviewed the therapeutic benefits of metformin and the details of its mechanism for a molecule of boon in various conditions like diabetes, prediabetes, obesity, polycystic ovarian disease, metabolic derangement in HIV, various cancers and aging.

Exposure of progressive immune dysfunction by SARS-CoV-2 mRNA vaccination in patients with chronic lymphocytic leukemia: A prospective cohort study.

BACKGROUND: Patients with chronic lymphocytic leukemia (CLL) have reduced seroconversion rates and lower binding antibody (Ab) and neutralizing antibody (NAb) titers than healthy individuals following Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) mRNA vaccination. Here, we dissected vaccine-mediated humoral and cellular responses to understand the mechanisms underlying CLL-induced immune dysfunction. METHODS AND FINDINGS: We performed a prospective observational study in SARS-CoV-2 infection-naïve CLL patients (n = 95) and healthy controls (n = 30) who were vaccinated between December 2020 and June 2021. Sixty-one CLL patients and 27 healthy controls received 2 doses of the Pfizer-BioNTech BNT162b2 vaccine, while 34 CLL patients and 3 healthy controls received 2 doses of the Moderna mRNA-1273 vaccine. The median time to analysis was 38 days (IQR, 27 to 83) for CLL patients and 36 days (IQR, 28 to 57) for healthy controls. Testing plasma samples for SARS-CoV-2 anti-spike and receptor-binding domain Abs by enzyme-linked immunosorbent assay (ELISA), we found that all healthy controls seroconverted to both antigens, while CLL patients had lower response rates (68% and 54%) as well as lower median titers (23-fold and 30-fold; both p < 0.001). Similarly, NAb responses against the then prevalent D614G and Delta SARS-CoV-2 variants were detected in 97% and 93% of controls, respectively, but in only 42% and 38% of CLL patients, who also exhibited >23-fold and >17-fold lower median NAb titers (both p < 0.001). Interestingly, 26% of CLL patients failed to develop NAbs but had high-titer binding Abs that preferentially reacted with the S2 subunit of the SARS-CoV-2 spike. Since these patients were also seropositive for endemic human coronaviruses (HCoVs), these responses likely reflect cross-reactive HCoV Abs rather than vaccine-induced de novo responses. CLL disease status, advanced Rai stage (III-IV), elevated serum beta-2 microglobulin levels (ß2m >2.4 mg/L), prior therapy, anti-CD20 immunotherapy (<12 months), and intravenous immunoglobulin (IVIg) prophylaxis were all predictive of an inability to mount SARS-CoV-2 NAbs (all p ≤ 0.03). T cell response rates determined for a subset of participants were 2.8-fold lower for CLL patients compared to healthy controls (0.05, 95% CI 0.01 to 0.27, p < 0.001), with reduced intracellular IFNγ staining (p = 0.03) and effector polyfunctionality (p < 0.001) observed in CD4+ but not in CD8+ T cells. Surprisingly, in treatment-naïve CLL patients, BNT162b2 vaccination was identified as an independent negative risk factor for NAb generation (5.8, 95% CI 1.6 to 27, p = 0.006). CLL patients who received mRNA-1273 had 12-fold higher (p < 0.001) NAb titers and 1.7-fold higher (6.5, 95% CI 1.3 to 32, p = 0.02) response rates than BNT162b2 vaccinees despite similar disease characteristics. The absence of detectable NAbs in CLL patients was associated with reduced naïve CD4+ T cells (p = 0.03) and increased CD8+ effector memory T cells (p = 0.006). Limitations of the study were that not all participants were subjected to the same immune analyses and that pre-vaccination samples were not available. CONCLUSIONS: CLL pathogenesis is characterized by a progressive loss of adaptive immune functions, including in most treatment-naïve patients, with preexisting memory being preserved longer than the capacity to mount responses to new antigens. In addition, higher NAb titers and response rates identify mRNA-1273 as a superior vaccine for CLL patients.

Potential of Cytochrome P450, a Family of Xenobiotic Metabolizing Enzymes, in Cancer Therapy.

Significance: Targeted cancer therapy with minimal off-target consequences has shown promise for some cancer types. Although cytochrome P450 (CYP) consists of 18 families, CYP1-4 families play key role in metabolizing xenobiotics and cancer drugs. This eventually affects the process of carcinogenesis, treatment outcomes, and cancer drug resistance. Differential overexpression of CYPs in transformed cells, together with phenotypic alterations in tumors, presents a potential for therapeutic intervention. Recent Advances: Recent advances in molecular tools and information technology have helped utilize CYPs as cancer targets. The precise expression in various tumors, X-ray crystal structures, improved understanding of the structure-activity relationship, and new approaches in the development of prodrugs have supported the ongoing efforts to develop CYP-based drugs with a better therapeutic index. Critical Issues: Narrow therapeutic index, off-target effects, drug resistance, and tumor heterogeneity limit the benefits of CYP-based conventional cancer therapies. In this review, we address the CYP1-4 families as druggable targets in cancer. An emphasis is given to the CYP expression, function, and the possible mechanisms that drive expression and activity in normal and transformed tissues. The strategies that inhibit or activate CYPs for therapeutic benefits are also discussed. Future Directions: Efforts are needed to develop more selective tools that will help comprehend molecular and metabolic alterations in tumor tissues with biological end-points in relation to CYPs. This will eventually translate to developing more specific CYP inhibitors/inducers. Antioxid. Redox Signal. 38, 853-876.

SARS-CoV-2 mRNA vaccination exposes progressive adaptive immune dysfunction in patients with chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) patients have lower seroconversion rates and antibody titers following SARS-CoV-2 vaccination, but the reasons for this diminished response are poorly understood. Here, we studied humoral and cellular responses in 95 CLL patients and 30 healthy controls after two BNT162b2 or mRNA-2173 mRNA immunizations. We found that 42% of CLL vaccinees developed SARS-CoV-2-specific binding and neutralizing antibodies (NAbs), while 32% had no response. Interestingly, 26% were seropositive, but had no detectable NAbs, suggesting the maintenance of pre-existing endemic human coronavirus-specific antibodies that cross-react with the S2 domain of the SARS-CoV-2 spike. These individuals had more advanced disease. In treatment-naive CLL patients, mRNA-2173 induced 12-fold higher NAb titers and 1.7-fold higher response rates than BNT162b2. These data reveal a graded loss of immune function, with pre-existing memory being preserved longer than the capacity to respond to new antigens, and identify mRNA-2173 as a superior vaccine for CLL patients.

Presentation of concurrent thrombotic thrombocytopenic purpura and Graves' disease.

Thrombotic thrombocytopenic purpura (TTP) is a type of thrombotic microangiopathy caused by deficient activity of ADAMTS13 that most commonly occurs secondary to an acquired autoantibody. There are limited data on the association between TTP and autoimmune thyroid disease. We present a case of acquired TTP in the setting of thyrotoxicosis from Graves' disease. Our patient improved with standard treatment of both TTP and thyrotoxicosis. A retrospective review of patients with TTP at our institution demonstrated that 32% had another autoimmune disorder, highlighting the concept of polyautoimmunity. These findings suggest an association between TTP and uncontrolled autoimmune disease. In patients with newly diagnosed TTP, physicians should evaluate for other autoimmune diseases and check thyroid function tests.

Online Strategies To Improve Quantitative Skills in Microbiology Laboratory Classes.

Biology is an increasingly quantitative science. Thus, it is important that undergraduate biology curricula include frequent opportunities for students to practice their quantitative skills. This can create a substantial grading burden for faculty teaching online and/or large enrollment courses, but the "formula question" feature present in many learning management systems (LMS) offers a solution. Using this feature, faculty set up a basic scaffold for an algebraic word problem, and the LMS can then automatically generate and grade many different versions of the question. In this paper, we describe the use of "formula questions" in an undergraduate microbiology course and specifically focus on how the strategic use of algebraic word problems at multiple points throughout the semester can help build quantitative literacy. Key to the success of this approach is that faculty provide a review of foundational mathematical skills early in the semester, even in upper-level classes. This should include reacquainting students with formatting conventions (e.g., rounding and scientific notation), familiarizing them with any idiosyncrasies of the technology platforms, and demonstrating how to solve math problems using spreadsheets. This initial effort increases student success when more complex problems are introduced later in the semester. Though the tips summarized in this paper focus on undergraduate microbiology teaching laboratories using Canvas, the approach can easily be modified to help students develop their critical thinking and quantitative reasoning skills at other levels and in other disciplines.

Cooperative microbial interactions mediate community biogeochemical responses to saltwater intrusion in wetland soils.

In freshwater wetlands, competitive and cooperative interactions between respiratory, fermentative and methanogenic microbes mediate the decomposition of organic matter. These interactions may be disrupted by saltwater intrusion disturbances that enhance the activity of sulfate-reducing bacteria (SRB), intensifying their competition with syntrophic bacteria and methanogens for electron donors. We simulated saltwater intrusion into wetland soil microcosms and examined biogeochemical and microbial responses, employing metabolic inhibitors to isolate the activity of various microbial functional groups. Sulfate additions increased total carbon dioxide production but decreased methane production. Butyrate degradation assays showed continued (but lower) levels of syntrophic metabolism despite strong demand by SRB for this key intermediate decomposition product and a shift in the methanogen community toward acetoclastic members. One month after removing SRB competition, total methane production recovered by only ∼50%. Similarly, butyrate assays showed an altered accumulation of products (including less methane), although overall rates of syntrophic butyrate breakdown largely recovered. These effects illustrate that changes in carbon mineralization following saltwater intrusion are driven by more than the oft-cited competition between SRB and methanogens for shared electron donors. Thus, the impacts of disturbances on wetland biogeochemistry are likely to persist until cooperative and competitive microbial metabolic interactions can recover fully.

Persistence of wastewater-associated antibiotic resistant bacteria in river microcosms.

The spread of antibiotic-resistant bacteria (ARB) associated with wastewater is a significant environmental concern, but little is known about the persistence and proliferation of these organisms in receiving water bodies after discharge. To address this knowledge gap, we performed a series of microcosm experiments in which river water was amended with either untreated or treated wastewater, and the abundance of viable ciprofloxacin-, Bactrim-, and erythromycin-resistant bacteria was monitored for 72 h. Both types of wastewater amendments significantly increased the initial abundance of ARB compared to microcosms containing only river water (all p < 0.03). The increase was greatest with untreated wastewater, but that effect decreased steadily over time. In contrast, microcosms amended with treated wastewater saw a smaller initial increase and more complex temporal dynamics. Following a brief lag, ARB abundance bloomed for all three of the antibiotics that we considered. This suggests that ARB that survive wastewater treatment are particularly hardy and may proliferate in riverine conditions after a short recovery period. To determine how interactions with the native river microbial community impacted the persistence of wastewater-associated ARB, an additional set of microcosms was prepared using filter-sterilized river water. Peak abundance in these microcosms was significantly higher by 1-2 orders of magnitude compared to microcosms containing an intact river microbial community (all p < 0.05), which suggests that biotic interactions play a significant role in regulating the persistence and proliferation of ARB. The data presented in this paper are among the first available that specifically consider persistence of viable ARB and represent an important step toward understanding AR-related human health risks downstream from wastewater discharge points and following sewer overflow events. Additional studies that consider longer time scales and the interplay of biotic and abiotic variables are essential for modeling public health risks associated with wastewater inputs of ARB to rivers and other aquatic environments.

Immunotherapy and Targeted Therapy in the Management of Oral Cancers.

Oral cancers (OCs), being one of the frequent malignancies in the head and neck region, need prompt diagnosis and treatment. Apart from basic therapeutic modalities, immunotherapy has now been utilized as a novel approach to combat the disease. With the comprehension of the strategies adopted by cancer cells to evade the immune elimination by the body's immune system, targeted immunotherapies have now become the core area of research. The immune expression of epidermal growth factor receptor (EGFR), programmed cell death protein ligand-1 (PDL-1), etc., are enhanced in OC and have been associated with evasion of the immune system. Targeted immunotherapies now include monoclonal antibodies targeting EGFR like cetuximab and panitumumab, programmed cell death-1 (PD-1) inhibitors like pembrolizumab, cemiplimab, and nivolumab, and PD-L1 inhibitors like atezolizumab, avelumab, and durvalumab. Targeted immunotherapies like chimeric antigen receptor T-cell treatment and small molecule inhibitors are in several clinical trials tried as monotherapy and adjuvant immunotherapy and have shown promising results. Other immunothera-peutic approaches such as cytokines like interferons or interleukins, vaccines, and gene therapy have also been an area of research for the management of OC. However, the cautious selection of appropriate patients with specific immune characteristics as a candidate for immunotherapeutic agents is a crucial component of targeted immunotherapy. This article elaborates on the immune contexture of oral cancer cells, the mechanism of immune evasion by cancer cells, targets for immunotherapies, existent immunotherapeutic agents, and prospects in the field of immunotherapy.

Phylogenetic organization in the assimilation of chemically distinct substrates by soil bacteria.

Soils are among the most biodiverse habitats on earth and while the species composition of microbial communities can influence decomposition rates and pathways, the functional significance of many microbial species and phylogenetic groups remains unknown. If bacteria exhibit phylogenetic organization in their function, this could enable ecologically meaningful classification of bacterial clades. Here, we show non-random phylogenetic organization in the rates of relative carbon assimilation for both rapidly mineralized substrates (amino acids and glucose) assimilated by many microbial taxa and slowly mineralized substrates (lipids and cellulose) assimilated by relatively few microbial taxa. When mapped onto bacterial phylogeny using ancestral character estimation this phylogenetic organization enabled the identification of clades involved in the decomposition of specific soil organic matter substrates. Phylogenetic organization in substrate assimilation could provide a basis for predicting the functional attributes of uncharacterized microbial taxa and understanding the significance of microbial community composition for soil organic matter decomposition.

SARS-CoV-2 mRNA vaccination exposes progressive adaptive immune dysfunction in patients with chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) patients have lower seroconversion rates and antibody titers following SARS-CoV-2 vaccination, but the reasons for this diminished response are poorly understood. Here, we studied humoral and cellular responses in 95 CLL patients and 30 healthy controls after two BNT162b2 or mRNA-2173 mRNA immunizations. We found that 42% of CLL vaccinees developed SARS-CoV-2-specific binding and neutralizing antibodies (NAbs), while 32% had no response. Interestingly, 26% were seropositive, but had no detectable NAbs, suggesting the maintenance of pre-existing endemic human coronavirus-specific antibodies that cross-react with the S2 domain of the SARS-CoV-2 spike. These individuals had more advanced disease. In treatment-naïve CLL patients, mRNA-2173 induced 12-fold higher NAb titers and 1.7-fold higher response rates than BNT162b2. These data reveal a graded loss of immune function, with pre-existing memory being preserved longer than the capacity to respond to new antigens, and identify mRNA-2173 as a superior vaccine for CLL patients.

Trace Metal Availability Affects Greenhouse Gas Emissions and Microbial Functional Group Abundance in Freshwater Wetland Sediments.

We investigated the effects of trace metal additions on microbial nitrogen (N) and carbon (C) cycling using freshwater wetland sediment microcosms amended with micromolar concentrations of copper (Cu), molybdenum (Mo), iron (Fe), and all combinations thereof. In addition to monitoring inorganic N transformations (NO3 -, NO2 -, N2O, NH4 +) and carbon mineralization (CO2, CH4), we tracked changes in functional gene abundance associated with denitrification (nirS, nirK, nosZ), dissimilatory nitrate reduction to ammonium (DNRA; nrfA), and methanogenesis (mcrA). With regards to N cycling, greater availability of Cu led to more complete denitrification (i.e., less N2O accumulation) and a higher abundance of the nirK and nosZ genes, which encode for Cu-dependent reductases. In contrast, we found sparse biochemical evidence of DNRA activity and no consistent effect of the trace metal additions on nrfA gene abundance. With regards to C mineralization, CO2 production was unaffected, but the amendments stimulated net CH4 production and Mo additions led to increased mcrA gene abundance. These findings demonstrate that trace metal effects on sediment microbial physiology can impact community-level function. We observed direct and indirect effects on both N and C biogeochemistry that resulted in increased production of greenhouse gasses, which may have been mediated through the documented changes in microbial community composition and shifts in functional group abundance. Overall, this work supports a more nuanced consideration of metal effects on environmental microbial communities that recognizes the key role that metal limitation plays in microbial physiology.

Authentic Modeling of Human Respiratory Virus Infection in Human Pluripotent Stem Cell-Derived Lung Organoids.

Infectious viruses so precisely fit their hosts that the study of natural viral infection depends on host-specific mechanisms that affect viral infection. For human parainfluenza virus 3, a prevalent cause of lower respiratory tract disease in infants, circulating human viruses are genetically different from viruses grown in standard laboratory conditions; the surface glycoproteins that mediate host cell entry on circulating viruses are suited to the environment of the human lung and differ from those of viruses grown in cultured cells. Polarized human airway epithelium cultures have been used to represent the large, proximal airways of mature adult airways. Here we modeled respiratory virus infections that occur in children or infect the distal lung using lung organoids that represent the entire developing infant lung. These 3D lung organoids derived from human pluripotent stem cells contain mesoderm and pulmonary endoderm and develop into branching airway and alveolar structures. Whole-genome sequencing analysis of parainfluenza viruses replicating in the organoids showed maintenance of nucleotide identity, suggesting that no selective pressure is exerted on the virus in this tissue. Infection with parainfluenza virus led to viral shedding without morphological changes, while respiratory syncytial virus infection induced detachment and shedding of infected cells into the lung organoid lumens, reminiscent of parainfluenza and respiratory syncytial virus in human infant lungs. Measles virus infection, in contrast, induced syncytium formation. These human stem cell-derived lung organoids may serve as an authentic model for respiratory viral pathogenesis in the developing or infant lung, recapitulating respiratory viral infection in the host.IMPORTANCE Respiratory viruses are among the first pathogens encountered by young children, and the significant impact of these viral infections on the developing lung is poorly understood. Circulating viruses are suited to the environment of the human lung and are different from those of viruses grown in cultured cells. We modeled respiratory virus infections that occur in children or infect the distal lung using lung organoids that represent the entire developing infant lung. These 3D lung organoids, derived from human pluripotent stem cells, develop into branching airway and alveolar structures and provide a tissue environment that maintains the authentic viral genome. The lung organoids can be genetically engineered prior to differentiation, thereby generating tissues bearing or lacking specific features that may be relevant to viral infection, a feature that may have utility for the study of host-pathogen interaction for a range of lung pathogens.

Wood Decay Characteristics and Interspecific Interactions Control Bacterial Community Succession in Populus grandidentata (Bigtooth Aspen).

Few studies have investigated bacterial community succession and the role of bacterial decomposition over a continuum of wood decay. Here, we identified how (i) the diversity and abundance of bacteria changed along a chronosequence of decay in Populus grandidentata (bigtooth aspen); (ii) bacterial community succession was dependent on the physical and chemical characteristics of the wood; (iii) interspecific bacterial interactions may mediate community structure. Four hundred and fifty-nine taxa were identified through Illumina sequencing of 16S rRNA amplicons from samples taken along a continuum of decay, representing standing dead trees, downed wood, and soil. Community diversity increased as decomposition progressed, peaking in the most decomposed trees. While a small proportion of taxa displayed a significant pattern in regards to decay status of the host log, many bacterial taxa followed a stochastic distribution. Changes in the water availability and chemical composition of standing dead and downed trees and soil were strongly coupled with shifts in bacterial communities. Nitrogen was a major driver of succession and nitrogen-fixing taxa of the order Rhizobiales were abundant early in decomposition. Recently downed logs shared 65% of their bacterial abundance with the microbiomes of standing dead trees while only sharing 16% with soil. As decay proceeds, bacterial communities appear to respond less to shifting resource availability and more to interspecific bacterial interactions - we report an increase in both the proportion (+9.3%) and the intensity (+62.3%) of interspecific interactions in later stages of decomposition, suggesting the emergence of a more complex community structure as wood decay progresses.

Novel microbial community composition and carbon biogeochemistry emerge over time following saltwater intrusion in wetlands.

Sea level rise and changes in precipitation can cause saltwater intrusion into historically freshwater wetlands, leading to shifts in microbial metabolism that alter greenhouse gas emissions and soil carbon sequestration. Saltwater intrusion modifies soil physicochemistry and can immediately affect microbial metabolism, but further alterations to biogeochemical processing can occur over time as microbial communities adapt to the changed environmental conditions. To assess temporal changes in microbial community composition and biogeochemical activity due to saltwater intrusion, soil cores were transplanted from a tidal freshwater marsh to a downstream mesohaline marsh and periodically sampled over 1 year. This experimental saltwater intrusion produced immediate changes in carbon mineralization rates, whereas shifts in the community composition developed more gradually. Salinity affected the composition of the prokaryotic community but did not exert a strong influence on the community composition of fungi. After only 1 week of saltwater exposure, carbon dioxide production doubled and methane production decreased by three orders of magnitude. By 1 month, carbon dioxide production in the transplant was comparable to the saltwater controls. Over time, we observed a partial recovery in methane production which strongly correlated with an increase in the relative abundance of three orders of hydrogenotrophic methanogens. Taken together, our results suggest that ecosystem responses to saltwater intrusion are dynamic over time as complex interactions develop between microbial communities and the soil organic carbon pool. The gradual changes in microbial community structure we observed suggest that previously freshwater wetlands may not experience an equilibration of ecosystem function until long after initial saltwater intrusion. Our results suggest that during this transitional period, likely lasting years to decades, these ecosystems may exhibit enhanced greenhouse gas production through greater soil respiration and continued methanogenesis.

Practice Patterns and Barriers for Intravenous Thrombolysis: A Survey of Neurologists in Saudi Arabia.

Only a small fraction of patients with acute ischemic stroke receive intravenous thrombolysis (IVT). We sought to assess barriers and practice patterns in using IVT for acute ischemic stroke among neurologists in Saudi Arabia. An electronic survey was sent to all neurologists registered with the Saudi Commission for Health Specialties. A total of 148 (77.5%) neurologists responded. The most common reported barriers for IVT administration were delayed presentation to hospitals (82.4%) and unclear time of symptom onset (50.0%). Only 9.9% of neurologists reported strict adherence to the American Heart Association/American Stroke Association guidelines for IVT administration. The most frequently waived criteria were "minor stroke with National Institutes of Health Stroke Scale [NIHSS] < 5" (49.4%) and "seizure at onset" (45.7%). For the extended 3-4.5-hour window, 18.5% of neurologists reported strict adherence to the four exclusion criteria. The most frequently waived criteria were "age older than 80 years" (53.1%) and "history of both diabetes and prior stroke" (42.0%). In conclusion, most neurologists do not adhere to the IVT exclusion criteria. However, little consensus exists regarding which criteria do not interfere with IVT administration. Barriers to IVT administration were identified and require immediate action by healthcare authorities in Saudi Arabia.

MinION™ nanopore sequencing of environmental metagenomes: a synthetic approach.

Environmental metagenomic analysis is typically accomplished by assigning taxonomy and/or function from whole genome sequencing or 16S amplicon sequences. Both of these approaches are limited, however, by read length, among other technical and biological factors. A nanopore-based sequencing platform, MinION™, produces reads that are ≥1 × 104 bp in length, potentially providing for more precise assignment, thereby alleviating some of the limitations inherent in determining metagenome composition from short reads. We tested the ability of sequence data produced by MinION (R7.3 flow cells) to correctly assign taxonomy in single bacterial species runs and in three types of low-complexity synthetic communities: a mixture of DNA using equal mass from four species, a community with one relatively rare (1%) and three abundant (33% each) components, and a mixture of genomic DNA from 20 bacterial strains of staggered representation. Taxonomic composition of the low-complexity communities was assessed by analyzing the MinION sequence data with three different bioinformatic approaches: Kraken, MG-RAST, and One Codex. Results: Long read sequences generated from libraries prepared from single strains using the version 5 kit and chemistry, run on the original MinION device, yielded as few as 224 to as many as 3497 bidirectional high-quality (2D) reads with an average overall study length of 6000 bp. For the single-strain analyses, assignment of reads to the correct genus by different methods ranged from 53.1% to 99.5%, assignment to the correct species ranged from 23.9% to 99.5%, and the majority of misassigned reads were to closely related organisms. A synthetic metagenome sequenced with the same setup yielded 714 high quality 2D reads of approximately 5500 bp that were up to 98% correctly assigned to the species level. Synthetic metagenome MinION libraries generated using version 6 kit and chemistry yielded from 899 to 3497 2D reads with lengths averaging 5700 bp with up to 98% assignment accuracy at the species level. The observed community proportions for "equal" and "rare" synthetic libraries were close to the known proportions, deviating from 0.1% to 10% across all tests. For a 20-species mock community with staggered contributions, a sequencing run detected all but 3 species (each included at <0.05% of DNA in the total mixture), 91% of reads were assigned to the correct species, 93% of reads were assigned to the correct genus, and >99% of reads were assigned to the correct family. Conclusions: At the current level of output and sequence quality (just under 4 × 103 2D reads for a synthetic metagenome), MinION sequencing followed by Kraken or One Codex analysis has the potential to provide rapid and accurate metagenomic analysis where the consortium is comprised of a limited number of taxa. Important considerations noted in this study included: high sensitivity of the MinION platform to the quality of input DNA, high variability of sequencing results across libraries and flow cells, and relatively small numbers of 2D reads per analysis limit. Together, these limited detection of very rare components of the microbial consortia, and would likely limit the utility of MinION for the sequencing of high-complexity metagenomic communities where thousands of taxa are expected. Furthermore, the limitations of the currently available data analysis tools suggest there is considerable room for improvement in the analytical approaches for the characterization of microbial communities using long reads. Nevertheless, the fact that the accurate taxonomic assignment of high-quality reads generated by MinION is approaching 99.5% and, in most cases, the inferred community structure mirrors the known proportions of a synthetic mixture warrants further exploration of practical application to environmental metagenomics as the platform continues to develop and improve. With further improvement in sequence throughput and error rate reduction, this platform shows great promise for precise real-time analysis of the composition and structure of more complex microbial communities.