Respiratory Microbiome Disruption and Risk for Ventilator-Associated Lower Respiratory Tract Infection.

BACKGROUND: Ventilator-associated lower respiratory tract infection (VA-LRTI) is common among critically ill patients and has been associated with increased morbidity and mortality. In acute critical illness, respiratory microbiome disruption indices (MDIs) have been shown to predict risk for VA-LRTI, but their utility beyond the first days of critical illness is unknown. We sought to characterize how MDIs previously shown to predict VA-LRTI at initiation of mechanical ventilation change with prolonged mechanical ventilation, and if they remain associated with VA-LRTI risk. METHODS: We developed a cohort of 83 subjects admitted to a long-term acute care hospital due to their prolonged dependence on mechanical ventilation; performed dense, longitudinal sampling of the lower respiratory tract, collecting 1066 specimens; and characterized the lower respiratory microbiome by 16S rRNA sequencing as well as total bacterial abundance by 16S rRNA quantitative polymerase chain reaction. RESULTS: Cross-sectional MDIs, including low Shannon diversity and high total bacterial abundance, were associated with risk for VA-LRTI, but associations had wide posterior credible intervals. Persistent lower respiratory microbiome disruption showed a more robust association with VA-LRTI risk, with each day of (base e) Shannon diversity <2.0 associated with a VA-LRTI odds ratio of 1.36 (95% credible interval, 1.10-1.72). The observed association was consistent across multiple clinical definitions of VA-LRTI. CONCLUSIONS: Cross-sectional MDIs have limited ability to discriminate VA-LRTI risk during prolonged mechanical ventilation, but persistent lower respiratory tract microbiome disruption, best characterized by consecutive days with low Shannon diversity, may identify a population at high risk for infection and may help target infection-prevention interventions.

Before the Bullets Fly: The Physician's Role in Preventing Firearm Injury.

Firearm injury is a disease that is disproportionately prevalent in the United States. When a bullet hits a human being, it brings together multiple structural determinants of health into one acute, life-changing event. Firearm injury can lead to long-term mental and physical challenges for individuals, families, and communities. Despite the impact of this disease, physicians often underestimate their role in not only treating but also preventing firearm injury. Physicians can intervene through screening, counseling, community engagement, and advocacy, and can mobilize the health care systems they serve to engage with injury prevention. Physicians also play a key role in expanding the knowledge base on firearm injury through much-needed research on the epidemiology, context, and outcomes of firearm injury. When we treat firearm injury as a disease, we can develop and implement interventions from the clinic to the statehouse that can curb profound harms. This work and these opportunities belong not only to emergency physicians and trauma surgeons, but to all fields that evaluate and assess patients over the life course.

Subglottic suction frequency and adverse ventilator-associated events during critical illness.

OBJECTIVE: Tracheal intubation and mechanical ventilation provide essential support for patients with respiratory failure, but the course of mechanical ventilation may be complicated by adverse ventilator-associated events (VAEs), which may or may not be associated with infection. We sought to understand how the frequency of subglottic suction, an indicator of the quantity of sputum produced by ventilated patients, relates to the onset of all VAEs and infection-associated VAEs. DESIGN: We performed a case-crossover study including 87 patients with VAEs, and we evaluated 848 days in the pre-VAE period at risk for a VAE. SETTING AND PARTICIPANTS: Critically ill patients were recruited from the medical intensive care unit of an academic medical center. METHODS: We used the number of as-needed subglottic suctioning events performed per calendar day to quantify sputum production, and we compared the immediate pre-VAE period to the preceding period. We used CDC surveillance definitions for VAE and to categorize whether events were infection associated or not. RESULTS: Sputum quantity measured by subglottic suction frequency is greater in the period immediately prior to VAE than in the preceding period. However, it does not discriminate well between infection-associated VAEs and VAEs without associated infection. CONCLUSIONS: Subglottic suction frequency may serve as a valuable marker of sputum quantity, and it is associated with risk of a VAE. However, our results require validation in a broader population of mechanically ventilated patients and intensive care settings.

Increased Firearm Injury During the COVID-19 Pandemic: A Hidden Urban Burden.

BACKGROUND: Public health measures were instituted to reduce COVID-19 spread. A decrease in total emergency department volume followed, but the impact on injury is unknown. With lockdown and social distancing potentially increasing domicile discord, we hypothesized that intentional injury increased during COVID-19, driven primarily by an increase in penetrating trauma. STUDY DESIGN: A retrospective review of acute adult patient care in an urban Level I trauma center assessed injury patterns. Presenting patient characteristics and diagnoses from 6 weeks pre to 10 weeks post statewide stay-at-home orders (March 16, 2020) were compared, as well as with 2015-2019. Subsets were defined by intentionality (intentional vs nonintentional) and mechanism of injury (blunt vs penetrating). Fisher exact and Wilcoxon tests were used to compare proportions and means. RESULTS: There were 357 trauma patients that presented pre stay-at-home order and 480 that presented post stay-at-home order. Pre and post groups demonstrated differences in sex (35.6% vs 27.9% female; p = 0.02), age (47.4 ± 22.1 years vs 42 ± 20.3 years; p = 0.009), and race (1.4% vs 2.3% Asian; 63.3% vs 68.3% Black; 30.5% vs 22.3% White; and 4.8% vs 7.1% other; p = 0.03). Post stay-at-home order mechanism of injury revealed more intentional injury (p = 0.0008). Decreases in nonintentional trauma after adoption of social isolation paralleled declines in daily emergency department visits. Compared with earlier years, 2020 demonstrated a significantly greater proportion of intentional violent injury during the peripandemic months, especially from firearms. CONCLUSIONS: Unprecedented social isolation policies to address COVID-19 were associated with increased intentional injury, especially gun violence. Meanwhile, emergency department and nonintentional trauma visits decreased. Pandemic-related public health measures should embrace intentional injury prevention and management strategies.

Impact of Nasopharyngeal Specimen Quality on SARS-CoV-2 Test Performance

BackgroundThe SARS-CoV-2 reverse-transcription polymerase chain reaction (RT-PCR) cycle of threshold (Ct) has been used to estimate quantitative viral load, with the goal of targeting isolation precautions for individuals with COVID-19 and guiding public health interventions. However, variability in specimen quality can alter the Ct values obtained from SARS-CoV-2 clinical assays. We sought to define how variable nasopharyngeal (NP) swab quality impacts clinical SARS-CoV-2 test sensitivity. MethodsWe performed amplification of a human gene target ({beta}-actin) in parallel with a clinical RT-PCR targeting the SARS-CoV-2 ORF1ab gene for 1311 NP specimens collected from patients with clinical concern for COVID-19. We evaluated the relationship between NP specimen quality, characterized by high Ct values for the human gene target {beta}-actin Ct, and the probability of SARS-CoV-2 detection via logistic regression, as well as the linear relationship between SARS-CoV-2 and {beta}-actin Ct. ResultsLow quality NP swabs are less likely to detect SARS-CoV-2 (odds ratio 0.654, 95%CI 0.523 to 0.802). We observed a positive linear relationship between SARS-CoV-2 and {beta}-actin Ct values (slope 0.169, 95%CI 0.092 to 0.247). COVID-19 disease severity was not associated with {beta}-actin Ct values. ConclusionsVariability in NP specimen quality accounts for significant differences in the sensitivity of clinical SARS-CoV-2 assays. If unrecognized, low quality NP specimens, which are characterized by a low level of amplifiable human DNA target, may limit the application of SARS-CoV-2 Ct values to direct infection control and public health interventions.

Designing Higher Resolution Self-Assembled 3D DNA Crystals via Strand Terminus Modifications.

DNA tensegrity triangles self-assemble into rhombohedral three-dimensional crystals via sticky ended cohesion. Crystals containing two-nucleotide (nt) sticky ends (GA:TC) have been reported previously, and those crystals diffracted to 4.9 Å at beamline NSLS-I-X25. Here, we analyze the effect of varying sticky end lengths and sequences as well as the impact of 5'- and 3'-phosphates on crystal formation and resolution. Tensegrity triangle motifs having 1-, 2-, 3-, and 4-nt sticky ends all form crystals. X-ray diffraction data from the same beamline reveal that the crystal resolution for a 1-nt sticky end (G:C) and a 3-nt sticky end (GAT:ATC) were 3.4 and 4.2 Å, respectively. Resolutions were determined from complete data sets in each case. We also conducted trials that examined every possible combination of 1-nucleotide and 2-nucleotide sticky-ended phosphorylated strands and successfully crystallized all 16 possible combinations of strands. We observed the position of the 5'-phosphate on either the crossover (1), helical (2), or central strand (3) affected the resolution of the self-assembled crystals for the 2-turn monomer (3.0 Å for 1-2P-3P) and 2-turn dimer sticky ended (4.1 Å for 1-2-3P) systems. We have also examined the impact of the identity of the base flanking the sticky ends as well as the use of 3'-phosphate. We conclude that crystal resolution is not a simple consequence of the thermodynamics of the direct nucleotide pairing interactions involved in molecular cohesion in this system.

Ultra-sensitive quasi-distributed temperature sensor based on an apodized fiber Bragg grating.

This work targets a remarkable quasi-distributed temperature sensor based on an apodized fiber Bragg grating. To achieve this, the mathematical formula for a proposed apodization function is carried out and tested. Then, an optimization parametric process required to achieve the remarkable accuracy that is based on coupled mode theory (CMT) is done. A detailed investigation for the side lobe analysis, which is a primary judgment factor, especially in quasi-distributed configuration, is investigated. A comparison between elite selection of apodization profiles (extracted from related literatures) and the proposed modified-Nuttal profile is carried out covering reflectivity peak, full width half maximum (FWHM), and side lobe analysis. The optimization process concludes that the proposed modified-Nuttal profile with a length (L) of 15 mm and refractive index modulation amplitude (Δn) of 1.4×10-4 is the optimum choice for single-stage and quasi-distributed temperature sensor networks. At previous values, the proposed profile achieves an acceptable reflectivity peak of 10-0.426 dB, acceptable FWHM of 0.0808 nm, lowest side lobe maximum (SL max) of 7.037×10-12 dB, lowest side lobe average (SL avg) of 3.883×10-12 dB, and lowest side lobe suppression ratio (SLSR) of 1.875×10-11 dB. These optimized characteristics lead to an accurate single-stage sensor with a temperature sensitivity of 0.0136 nm/°C. For the quasi-distributed scenario, a noteworthy total isolation of 91 dB is achieved without temperature, and an isolation of 4.83 dB is achieved while applying temperature of 110°C for a five-stage temperature-sensing network. Further investigation is made proving that consistency in choosing the apodization profile in the quasi-distributed network is mandatory. If the consistency condition is violated, the proposed profile still survives with a casualty of side lobe level rise of -73.2070 dB when adding uniform apodization and -46.4823 dB when adding Gaussian apodization to the five-stage modified-Nuttall temperature-sensing network.

Therapeutic effect of carnosine in rat model of experimental liver carcinogenesis.

The possible anticancer effect of carnosine versus doxorubicin was investigated against hepatocellular carcinoma (HCC) induced by trichloroacetic acid (TCA) (500mg/kg/day, p.o., for 5days) in rats. Following induction of HCC, rats treated with either carnosine (10mg/kg/day, i.p.), or doxorubicin (2.5mg/kg, i.p., once weekly), for 2 weeks. Carnosine significantly decreased serum alanine aminotransferase, and hepatic lipid peroxidation, nitric oxide, tumor necrosis factor-α, and nuclear factor-κB p65 unit, and significantly increased liver total antioxidant status in TCA-challenged rats. The effects of doxorubicin on oxidative, nitrative, and inflammatory biomarkers were less significant than carnosine. However, both carnosine and doxorubicin significantly induced liver tissue apoptotic biomarkers, Bax, cytosolic cytochrome C, and caspase-3, in a comparable manner. Additionally, carnosine and doxorubicin reduced the histopathological dysplastic changes, and alpha-fetoprotein expression in liver of rats with HCC. It was concluded that carnosine significantly protected against TCA-induced liver carcinogenesis in rats, through its antioxidant, antinitrative, and anti-inflammatory effects, and induction of apoptosis.

Epigallocatechin-3-gallate counters cisplatin toxicity of rat testes.

CONTEXT: Epigallocatechin-3-gallate (EG), the main active flavonoid in green tea, has well-known anti-inflammatory, antioxidant, and anti-apoptotic activities. OBJECTIVE: The EG protection against testicular injury induced by cisplatin was studied in Sprague-Dawley rats. MATERIALS AND METHODS: Cisplatin (10 mg/kg, i.p) was given as a single injection to rats. EG was given at 40 and 80 mg/kg/day, i.p., for 5 days, starting the same day of cisplatin insult. Serum testosterone, and testicular malondialdehyde, total antioxidant status, nitric oxide, interleukin-6, interleukin-1ß, cytochrome C, Bax/Bcl-2 ratio, and caspase-3 were measured. In addition, testicular histopathological examination and immunohistochemical expression of testicular tumour necrosis factor-α were evaluated. RESULTS: Cisplatin, compared to the control, significantly decreased serum testosterone (6.48 ± 0.7 vs. 50.8 ± 4.91 ng/10 mL), and testicular tissue antioxidant status (17.3 ± 1.21 vs. 64.12 ± 5.4 µmol/g), and significantly increased interleukin-6 (85.81 ± 6.11 vs. 38.2 ± 2.79 pg/100 mg), interleukin-1ß (98.09 ± 8.31 vs. 32.52 ± 2.08 pg/100 mg), malondialdehyde (74.5 ± 5.88 vs. 23.8 ± 1.91 nmol/g), nitric oxide (104.98 ± 8.5 vs. 52.68 ± 5.12 nmol/100 mg), cytochrome C (5.97 ± 0.33 vs. 1.6 ± 0.99 ng/mg protein), Bax/Bcl-2 ratio (4.01 ± 0.38 vs. 0.71 ± 0.0), and caspase-3 (3.2 ± 0.21 vs. 0.98 ± 0.08 O.D. 405 nm) in rat testes. EG (40 and 80 mg/kg, respectively) caused significant increases of serum testosterone (33.9 ± 2.89 and 47.88 ± 4.4 ng/10 mL), and testicular antioxidant status (47.1 ± 3.92 and 58.22 ± 3.58 µmol/g), and significant decreases of interleukin-6 (57.39 ± 4.2 and 48.18 ± 3.98 pg/100 mg), interleukin-1ß (65.12 ± 5.88 and 41.96 ± 3.51 pg/100 mg), malondialdehyde (42.3 ± 3.9 and 28.67 ± 2.49 nmol/g), nitric oxide (70.6 ± 6.79 and 61.31 ± 5.18 nmol/100 mg), cytochrome C (3.4 ± 0.27 and 2.21 ± 0.18 ng/mg protein), Bax/Bcl-2 ratio (1.49 ± 0.14 and 1.1 ± 0.09), and caspase-3 (2.1 ± 0.17 and 1.48 ± 0.13 O.D. 405 nm) in testes of cisplatin-treated rats. Additionally, both doses of EG significantly ameliorated the histopathological injury and reduced tumour necrosis factor-α expression in rat testes. CONCLUSION: EG can afford testicular protection in cisplatin-challenged rats by its antioxidant, antinitrative, anti-inflammatory and antiapoptotic effects.

Identification, expansion and characterization of cancer cells with stem cell properties from head and neck squamous cell carcinomas.

Head and neck squamous cell carcinoma (HNSCC) is a major public health concern. Recent data indicate the presence of cancer stem cells (CSC) in many solid tumors, including HNSCC. Here, we assessed the stem cell (SC) characteristics, including cell surface markers, radioresistance, chromosomal instability, and in vivo tumorigenic capacity of CSC isolated from HNSCC patient specimens. We show that spheroid enrichment of CSC from early and short-term HNSCC cell cultures was associated with increased expression of CD44, CD133, SOX2 and BMI1 compared with normal oral epithelial cells. On immunophenotyping, five of 12 SC/CSC markers were homogenously expressed in all tumor cultures, while one of 12 was negative, four of 12 showed variable expression, and two of the 12 were expressed heterogeneously. We showed that irradiated CSCs survived and retained their self-renewal capacity across different ionizing radiation (IR) regimens. Fluorescence in situ hybridization (FISH) analyses of parental and clonally-derived tumor cells revealed different chromosome copy numbers from cell to cell, suggesting the presence of chromosomal instability in HNSCC CSC. Further, our in vitro and in vivo mouse engraftment studies suggest that CD44+/CD66- is a promising, consistent biomarker combination for HNSCC CSC. Overall, our findings add further evidence to the proposed role of HNSCC CSCs in therapeutic resistance.

Construction and Structure Determination of a Three-Dimensional DNA Crystal.

Structural DNA nanotechnology combines branched DNA junctions with sticky-ended cohesion to create self-assembling macromolecular architectures. One of the key goals of structural DNA nanotechnology is to construct three-dimensional (3D) crystalline lattices. Here we present a new DNA motif and a strategy that has led to the assembly of a 3D lattice. We have determined the X-ray crystal structures of two related constructs to 3.1 Å resolution using bromine-derivatized crystals. The motif we used employs a five-nucleotide repeating sequence that weaves through a series of two-turn DNA duplexes. The duplexes are tied into a layered structure that is organized and dictated by a concert of four-arm junctions; these in turn assemble into continuous arrays facilitated by sequence-specific sticky-ended cohesion. The 3D X-ray structure of these DNA crystals holds promise for the design of new structural motifs to create programmable 3D DNA lattices with atomic spatial resolution. The two arrays differ by the use of four or six repeats of the five-nucleotide units in the repeating but statistically disordered central strand. In addition, we report a 2D rhombuslike array formed from similar components.

Nephroprotection of punicalagin in rat model of endotoxemic acute kidney injury.

The potential nephroprotection of punicalagin (PNG) against lipopolysaccharide (LPS)-induced acute kidney injury in rats was investigated. Rats received a single i.v. dose of LPS (5 mg/kg), and treated with PNG (50 mg/kg, i.p.), 1 h before, and 1 h following LPS administration. LPS caused significant increases of serum creatinine and neutrophil gelatinase-associated lipocalin. LPS also resulted in significant increases in interleukin-18, tumor necrosis factor-α, interleukin-6, malondialdehyde, nitric oxide, Bax/Bcl-2 ratio and myeloperoxidase, inducible nitric oxide synthase, caspases 3, 8 and 9 activities, and a significant decrease in total antioxidant capacity in kidney tissues. PNG significantly ameliorated the alterations in the measured parameters. Additionally, PNG attenuated the histopathological injury and reduced kidney injury molecule-1 expression in kidneys of rats that received LPS. It was concluded that PNG ameliorated endotoxemic acute kidney injury in rats by counteracting inflammation, oxidative/nitrative stress and apoptosis.

Punicalagin alleviates hepatotoxicity in rats challenged with cyclophosphamide.

This study investigated the possible hepatoprotection of punicalagin in rats received cyclophosphamide (20mg/kg/day, i.p., for 7 days). Punicalagin given at two doses, 15 and 30mg/kg/day, p.o., for 7 days, starting the same day of cyclophosphamide administration. Punicalagin significantly and dose-dependently reduced the elevations of serum alanine aminotransferase, and liver nuclear factor-κB p65, tumor necrosis factor-α, interleukin-1ß, malondialdehyde, nitric oxide, Bax/Bcl-2 ratio, inducible nitric oxide synthase, caspases 3 and 9 activities, and prevented the decrease of hepatic total antioxidant capacity. Punicalagin also attenuated the histopathological liver tissue damage, and decreased cyclooxygenase-2 expression in liver of rats received cyclophosphamide in a dose-dependent manner. It was concluded that punicalagin protected rat liver against cyclophosphamide toxicity by inhibiting oxidative/nitrosative stress, inflammation, and apoptosis.

Stabilisation of self-assembled DNA crystals by triplex-directed photo-cross-linking.

The tensegrity triangle is a robust DNA motif that can self-assemble to generate macroscopic three-dimensional crystals. However, the stability of these crystals is dependent on the high ionic conditions used for crystal growth. Here we demonstrate that a triplex-forming oligonucleotide can be used to direct the specific intercalation, and subsequent photo-cross-linking, of 4,5',8-trimethylpsoralen to single or multiple loci within or between the tiles of the crystal. Cross-linking between the tiles of the crystal improves their thermal stability. Such an approach is likely to facilitate the removal of crystals from their mother liquor and may prove useful for applications that require greater crystal stability.

Cell division patterns and chromosomal segregation defects in oral cancer stem cells.

Oral squamous cell carcinoma (OSCC) is a serious public health problem caused primarily by smoking and alcohol consumption or human papillomavirus. The cancer stem cell (CSC) theory posits that CSCs show unique characteristics, including self-renewal and therapeutic resistance. Examining biomarkers and other features of CSCs is critical to better understanding their biology. To this end, the results show that cellular SOX2 immunostaining correlates with other CSC biomarkers in OSCC cell lines and marks the rare CSC population. To assess whether CSC division patterns are symmetrical, resulting in two CSC, or asymmetrical, leading to one CSC and one cancer cell, cell size and fluorescence intensity of mitotic cells stained with SOX2 were analyzed. Asymmetrical SOX2 distribution in ≈25% of the mitoses analyzed was detected. Chromosomal instability, some of which is caused by chromosome segregation defects (CSDs), is a feature of cancer cells that leads to altered gene copy numbers. We compare chromosomal instability (as measured by CSDs) between CSCs (SOX2+) and non-CSCs (SOX2-) from the same OSCC cell lines. CSDs were more common in non-CSCs (SOX2-) than CSCs (SOX2+) and in symmetrical CSC (SOX2+) mitotic pairs than asymmetrical CSC (SOX2+/SOX2-) mitotic pairs. CSCs showed fewer and different types of CSDs after ionizing radiation treatment than non-CSCs. Overall, these data are the first to demonstrate both symmetrical and asymmetrical cell divisions with CSDs in OSCC CSC. Further, the results suggest that CSCs may undergo altered behavior, including therapeutic resistance as a result of chromosomal instability due to chromosome segregation defects. © 2016 Wiley Periodicals, Inc.

Covalent Linkage of One-Dimensional DNA Arrays Bonded by Paranemic Cohesion.

The construction of DNA nanostructures from branched DNA motifs, or tiles, typically relies on the use of sticky-ended cohesion, owing to the specificity and programmability of DNA sequences. The stability of such constructs when unligated is restricted to a specific range of temperatures, owing to the disruption of base pairing at elevated temperatures. Paranemic (PX) cohesion was developed as an alternative to sticky ends for the cohesion of large topologically closed species that could be purified reliably on denaturing gels. However, PX cohesion is also of limited stability. In this work, we added sticky-ended interactions to PX-cohesive complexes to create interlocked complexes by functionalizing the sticky ends with psoralen, which can form cross-links between the two strands of a double helix. We were able to reinforce the stability of the constructs by creating covalent linkages between the 3'-ends and 5'-ends of the sticky ends; the sticky ends were added to double crossover domains via 3'-3' and 5'-5' linkages. Catenated arrays were obtained either by enzymatic ligation or by UV cross-linking. We have constructed finite-length one-dimensional arrays linked by interlocking loops and have positioned streptavidin-gold particles on these constructs.

Topological Linkage of DNA Tiles Bonded by Paranemic Cohesion.

Catenation is the process by which cyclic strands are combined like the links of a chain, whereas knotting changes the linking properties of a single strand. In the cell, topoisomerases catalyzing strand passage operations enable the knotting and catenation of DNA so that single- or double-stranded segments can be passed through each other. Here, we use a system of closed DNA structures involving a paranemic motif, called PX-DNA, to bind double strands of DNA together. These PX-cohesive closed molecules contain complementary loops whose linking by Escherichia coli topoisomerase 1 (Topo 1) leads to various types of catenated and knotted structures. We were able to obtain specific DNA topological constructs by varying the lengths of the complementary tracts between the complementary loops. The formation of the structures was analyzed by denaturing gel electrophoresis, and the various topologies of the constructs were characterized using the program Knotilus.

Fat, fibre and cancer risk in African Americans and rural Africans.

Rates of colon cancer are much higher in African Americans (65:100,000) than in rural South Africans (<5:100,000). The higher rates are associated with higher animal protein and fat, and lower fibre consumption, higher colonic secondary bile acids, lower colonic short-chain fatty acid quantities and higher mucosal proliferative biomarkers of cancer risk in otherwise healthy middle-aged volunteers. Here we investigate further the role of fat and fibre in this association. We performed 2-week food exchanges in subjects from the same populations, where African Americans were fed a high-fibre, low-fat African-style diet and rural Africans a high-fat, low-fibre western-style diet, under close supervision. In comparison with their usual diets, the food changes resulted in remarkable reciprocal changes in mucosal biomarkers of cancer risk and in aspects of the microbiota and metabolome known to affect cancer risk, best illustrated by increased saccharolytic fermentation and butyrogenesis, and suppressed secondary bile acid synthesis in the African Americans.