Microbiome analyses of poultry feeds: Part II. Comparison of different poultry feeds.

Within the realm of poultry feed mill operations, the persistent concern over microbial feed quality necessitates the establishment of a robust baseline for enhancing and sustaining the standards of commercial feeds. This dual-phase investigation, comprising Parts I, was previously published, and the current study presented here as Part II aimed to illuminate this baseline using 16S rRNA gene sequencing. In Part II, nine distinct commercial poultry feeds formulated as starters, growers, starter/growers, or supplements, the selected feeds underwent genomic DNA extraction, amplification with custom dual-indexed primers, and subsequent Illumina MiSeq sequencing. Through data analysis in QIIME2-2021.4 and R Studio, the study unveils alpha (Kruskal-Wallis) and beta (ANOSIM) diversity, taxonomic differences (ANCOM), and core microbiomes (core_members), deeming main and pairwise effects statistically significant at p < 0.05 and Q < 0.05. Notably, the investigation identified 30% common core microbial members across the nine feed types, shedding light on potential foodborne poultry pathogens such as Helicobacter and Campylobacter. Probiotic-associated feeds exhibited distinct microbial communities, emphasizing the need to explore their impact on the early poultry gastrointestinal tract (GIT) further.

Microbiome analyses of poultry feeds: Part I. Comparison of five different DNA extraction methods.

Given extensive variability in feed composition, the absence of a dedicated DNA extraction kit for poultry feed underscores the need for an optimized extraction technique for reliable downstream sequencing analyses. This study investigates the impact of five DNA extraction techniques: Qiagen QIAamp DNA Stool Mini Kit (Qiagen), modified Qiagen with Lysing Matrix B (MQ), modified Qiagen with celite purification (MQC), polyethylene glycol (PEG), and 1-Day Direct. Genomic DNA amplification and Illumina MiSeq sequencing were conducted. QIIME2-2021.4 facilitated data analysis, revealing significant diversity and compositional differences influenced by extraction methods. Qiagen exhibited lower evenness and richness compared to other methods. 1-Day Direct and PEG enhanced bacterial diversities by employing bead beating and lysozyme. Despite similar taxonomic resolution, the Qiagen kit provides a rapid, consistent method for assessing poultry feed microbiomes. Modified techniques (MQ and MQC) improve DNA purification, reducing bias in commercial poultry feed samples. PEG and 1-Day Direct methods were effective but may require standardization. Overall, this study underscores the importance of optimized extraction techniques in poultry feed analysis, with potential implications for future standardization of effective methods.

Rotating night shift work and menopausal age.

STUDY QUESTION: How are rotating night shift schedules associated with age at menopause among a large, national cohort of shift working nurses? SUMMARY ANSWER: Our findings suggest that working rotating night shifts with sufficient frequency may modestly accelerate reproductive senescence among women who may already be predisposed to earlier menopause. WHAT IS KNOWN ALREADY: Younger age at menopause has been associated with increased risk of adverse health outcomes, particularly those linked to reproduction. Night work has been associated with reproductive dysfunction, including disruption of menstrual cycle patterns. STUDY DESIGN, SIZE, DURATION: This cohort study was conducted among 80 840 women of the Nurses' Health Study 2 (NHS2), with prospective follow-up from 1991 through 2013. Loss-to-follow-up of the NHS2 is estimated to be <10%. PARTICIPANTS/MATERIALS, SETTING, METHODS: We assessed the association between cumulative and current rotating night shift work and age at natural menopause over 22 years of follow-up (1991-2013). Cox proportional hazards models were used to estimate hazard ratios (HR) for menopause, adjusted for age, smoking status, body mass index, physical activity, alcohol consumption, reproductive factors and exogenous hormone use. MAIN RESULTS AND THE ROLE OF CHANCE: Over follow-up, 27 456 women (34%) reached natural menopause. Women who worked 20 or more months of rotating night shifts in the prior 2-year had an increased risk of earlier menopause (multivariable-adjusted (MV)-HR = 1.09, 95% CI: 1.02-1.16) compared to women without rotating night shift work. This risk was stronger among women undergoing menopause or otherwise censored under age 45 years (MV-HR = 1.25, 95% CI: 1.08-1.46), than it was for those continuing in the study when >45 years old (MV-HR = 1.05, 95% CI: 0.99-1.13). Working 10 or more years of cumulative rotating night work was also associated with higher risk of menopause among women reaching menopause under age 45 (MV-HR10-19 years = 1.22, 95% CI: 1.03-1.44; MV-HR≥20 years = 1.73, 95% CI: 0.90-3.35), though not over the age of 45 years (MV-HR10-19 years = 1.04, 95% CI: 0.99-1.10; MV-HR≥20 years = 1.01, 95% CI: 0.89-1.15). LIMITATIONS, REASONS FOR CAUTION: The degree to which observed effects of rotating night shifts on age at natural menopause are due to circadian disruption, rather than fatigue and stress associated with working more demanding schedules, is uncertain due to potential residual confounding by these factors. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to assess the effects of night work on menopausal timing among a larger national cohort of shift working women. Women already prone to earlier menopause may further truncate their reproductive lifetime by working schedules comprising day as well as night shifts. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Center for Disease Control and Prevention/The National Institute for Occupational Safety and Health Grant 5R01OH009803 (PI: Schernhammer E), as well as UM1 CA176726 from the National Institute of Health. The funding sources had no role in the design and conduct of the study; collection, management, analysis and interpretation of the data; preparation, review or approval of the article; and decision to submit the article for publication. The authors have no conflicts of interest.

Multiple Structural Transitions in Langmuir Monolayers of Charged Soft-Shell Nanoparticles.

We have investigated the morphologies of Langmuir layers of charged, polymeric hard-core/interlayer/soft-shell nanoparticles spread at the air-water interface. Depending on various mutual interactions, which are correlated to the areal densities of the deposited nanoparticles, we observed ordered patterns of nondense and closed-packed arrangements of core/interlayer/shell (CIS) nanoparticle ordering. At low areal densities, we found an almost regular distribution of the charged CIS nanoparticles on the water surface, which resulted from long-range repulsive electrostatic interactions between them. At higher areal densities, domains of more closely packed and ordered nanoparticles were formed, coexisting with regions of randomly and sparsely distributed nanoparticles. We relate these domains to the interplay of electrostatic repulsion and capillary attraction caused by the dipolar character of like-charged particles at the interface, allowing for a characteristic separation distance between nanoparticles of about 3-4 times the nanoparticle diameter. At relatively high areal densities, attractive van der Waals forces were finally capable of making nanoparticles to come in contact with each other, leading to densely packed patches of hexagonally ordered nanoparticles coexisting with regions of rather well-ordered nanoparticles separated by about 1 µm and regions of randomly and sparsely distributed nanoparticles. Intriguingly, upon re-expansion of the area available per nanoparticle, these densely packed patches disappeared, indicating that steric repulsion due to the presence of soft shells as well as long-range electrostatic repulsive forces were strong enough to assure reversibility of the morphological behavior.

A centrally generated primary care physician audit report does not improve colonoscopy uptake after a positive result on a fecal occult blood test in Ontario's ColonCancerCheck program.

BACKGROUND: Timely follow-up of fecal occult blood screening with colonoscopy is essential for achieving colorectal cancer mortality reduction. In the present study, we evaluated the effectiveness of centrally generated, physician-targeted audit and feedback to improve colonoscopy uptake after a positive fecal occult blood test (fobt) result within Ontario's population-wide ColonCancerCheck Program. METHODS: This prospective cohort study used data sets from Ontario's ColonCancerCheck Program (2008-2011) that were linked to provincial administrative health databases. Cox proportional hazards regression was used to estimate the effect of centralized, physician-targeted audit and feedback on colonoscopy uptake in an Ontario-wide fobt-positive cohort. RESULTS: A mailed physician audit and feedback report identifying individuals outstanding for colonoscopy for 3 or more months after a positive fobt result did not increase the likelihood of colonoscopy uptake (hazard ratio: 0.95; 95% confidence interval: 0.79 to 1.13). Duration of positive fobt status was strongly inversely associated with the hazard of follow-up colonoscopy (p for linear trend: <0.001). CONCLUSIONS: In a large population-wide setting, centralized tracking in the form of physician-targeted mailed audit and feedback reports does not improve colonoscopy uptake for screening participants with a positive fobt result outstanding for 3 or more months. Mailed physician-targeted screening audit and feedback reports alone are unlikely to improve compliance with follow-up colonoscopy in Ontario. Other interventions such as physician audits or automatic referrals, demonstrated to be effective in other jurisdictions, might be warranted.

Evidence from 18S ribosomal RNA sequences that lampreys and hagfishes form a natural group.

Lampreys and hagfishes (cyclostomes) traditionally were considered to be a natural (monophyletic) group. Recently, the consensus of opinion, based largely on morphological analyses, has shifted to a view that lampreys are more closely related to jawed vertebrates (gnathostomes) than to hagfishes. Phylogenetic comparisons of 18S ribosomal RNA sequences from two hagfishes, two lampreys, a tunicate, a lancelet, and a number of gnathostomes support the monophyly of the cyclostomes. These data force a reassessment of several features of early vertebrate evolution.

Molecular architecture of the rotary motor in ATP synthase.

Adenosine triphosphate (ATP) synthase contains a rotary motor involved in biological energy conversion. Its membrane-embedded F0 sector has a rotation generator fueled by the proton-motive force, which provides the energy required for the synthesis of ATP by the F1 domain. An electron density map obtained from crystals of a subcomplex of yeast mitochondrial ATP synthase shows a ring of 10 c subunits. Each c subunit forms an alpha-helical hairpin. The interhelical loops of six to seven of the c subunits are in close contact with the gamma and delta subunits of the central stalk. The extensive contact between the c ring and the stalk suggests that they may rotate as an ensemble during catalysis.

Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 A resolution.

The three-dimensional structure of the proteasome from the archaebacterium Thermoplasma acidophilum has been elucidated by x-ray crystallographic analysis by means of isomorphous replacement and cyclic averaging. The atomic model was built and refined to a crystallographic R factor of 22.1 percent. The 673-kilodalton protease complex consists of 14 copies of two different subunits, alpha and beta, forming a barrel-shaped structure of four stacked rings. The two inner rings consist of seven beta subunits each, and the two outer rings consist of seven alpha subunits each. A narrow channel controls access to the three inner compartments. The alpha 7 beta 7 beta 7 alpha 7 subunit assembly has 72-point group symmetry. The structures of the alpha and beta subunits are similar, consisting of a core of two antiparallel beta sheets that is flanked by alpha helices on both sides. The binding of a peptide aldehyde inhibitor marks the active site in the central cavity at the amino termini of the beta subunits and suggests a novel proteolytic mechanism.

Proteasome from Thermoplasma acidophilum: a threonine protease.

The catalytic mechanism of the 20S proteasome from the archaebacterium Thermoplasma acidophilum has been analyzed by site-directed mutagenesis of the beta subunit and by inhibitor studies. Deletion of the amino-terminal threonine or its mutation to alanine led to inactivation of the enzyme. Mutation of the residue to serine led to a fully active enzyme, which was over ten times more sensitive to the serine protease inhibitor 3,4-dichloroisocoumarin. In combination with the crystal structure of a proteasome-inhibitor complex, the data show that the nucleophilic attack is mediated by the amino-terminal threonine of processed beta subunits. The conservation pattern of this residue in eukaryotic sequences suggests that at least three of the seven eukaryotic beta-type subunit branches should be proteolytically inactive.

The rotary mechanism of ATP synthase.

Since the chemiosmotic theory was proposed by Peter Mitchell in the 1960s, a major objective has been to elucidate the mechanism of coupling of the transmembrane proton motive force, created by respiration or photosynthesis, to the synthesis of ATP from ADP and inorganic phosphate. Recently, significant progress has been made towards establishing the complete structure of ATP synthase and revealing its mechanism. The X-ray structure of the F(1) catalytic domain has been completed and an electron density map of the F(1)-c(10) subcomplex has provided a glimpse of the motor in the membrane domain. Direct microscopic observation of rotation has been extended to F(1)-ATPase and F(1)F(o)-ATPase complexes.

Preliminary X-ray crystallographic study of the proteasome from Thermoplasma acidophilum.

Single cystals of proteasomes from the archaebacterium Thermoplasma acidophilum were obtained using the hanging-drop vapor diffusion method. The crystals diffract to better than 3.0 A and belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 308.9 A, b = 208.8 A and c = 116.9 A. There is one molecular complex in the asymmetric unit. Two potentially useful heavy-atom derivatives have been obtained. The self-rotation function of the native Patterson map shows local sevenfold symmetry, consistent with the low-resolution structure obtained by electron microscopic techniques. The unit cell dimensions and crystal symmetry together with the shape and size of the proteasome suggest a packing arrangement of proteasome molecules in the unit cell, with their cylinder axis nearly parallel to the crystallographic a-axis.

Crystal structure of the factor for inversion stimulation FIS at 2.0 A resolution.

The factor for inversion stimulation (FIS) binds as a homodimeric molecule to a loose 15 nucleotide consensus sequence in DNA. It stimulates DNA-related processes, such as DNA inversion and excision, it activates transcription of tRNA and rRNA genes and it regulates its own synthesis. FIS crystallizes as a homodimer, with 2 x 98 amino acid residues in the asymmetric unit. The crystal structure was determined with multiple isomorphous replacement and refined to an R-factor of 19.2% against all the 12,719 X-ray data (no sigma-cutoff) extending to 2.0 A resolution. The two monomers are related by a non-crystallographic dyad axis. The structure of the dimer is modular, with the first 23 amino acid residues in molecule M1 and the first 24 in molecule M2 disordered and not "seen" in the electron density. The polypeptide folds into four alpha-helices, with alpha A, alpha A' (amino acid residues 26 to 40) and alpha B, alpha B' (49 to 69) forming the core of the FIS dimer, which is stabilized by hydrophobic forces. To the core are attached "classical" helix-turn-helix motifs, alpha C, alpha D (73 to 81 and 84 to 94) and alpha C', alpha D'. The connections linking the helices are structured by two beta-turns for alpha A/alpha B, and alpha C1 type extensions are observed at the C termini of helices alpha B, alpha C and alpha D. Helices alpha D and alpha D' contain 2 x 6 positive charges; they are separated by 24 A and can bind adjacent major grooves in B-type DNA if it is bent 90 degrees. The modular structure of FIS is also reflected by mutation experiments; mutations in the N-terminal part and alpha A interfere with FIS binding to invertases, and mutations in the helix-turn-helix motif interfere with DNA binding.

Proteasome: from structure to function.

During the past two years, significant progress has been made in understanding the structure and function of the proteasome. Recent work has revealed the three-dimensional structure of the 700 kDa proteolytic complex at atomic resolution and elucidated its novel catalytic mechanism. Close relationships to a number of other amino-terminal hydrolases have emerged, making the proteasomal subunits the prototype of this newly discovered structural superfamily.

Consistency in the observation of features used to classify duct carcinoma in situ (DCIS) of the breast.

AIM: To determine interobserver and intra-observer agreement in the assessment of cytological grade and intraduct necrosis in pure duct carcinoma in situ (DCIS) of the breast. METHODS: Sixty unselected cases with illustrated diagnostic criteria were circulated to 19 practising histopathologists. RESULTS: Overall agreement was moderate for cytological grade in three categories: 71% agreement; weighted kappa (kappa w), 0.36; intraduct necrosis in three categories (absent, present, extensive): 76% agreement; kappa w, 0.57; and the Van Nuys classification system: 73% agreement; kappa w, 0.48. Agreement was no better among observers participating in the National External Quality Assurance Programme. Intra-observer agreement for cytological assessment (69.6% agreement; kappa w, 0.52) and intraduct necrosis (68.3% agreement; kappa w, 0.48) was moderate, suggesting that individual variation rather than precision of criteria contributes to the lack of agreement. CONCLUSIONS: Moderate agreement on observations can be achieved by non-specialist pathologists, with better agreement on necrosis than cytological grade. There was evidence of consistent individual bias towards over or under scoring cytological grade, which could be corrected with adequate and prompt feedback.

Cardiovascular effects of beta-carbolines in conscious rats.

The beta-carbolines have a high affinity for the benzodiazepine receptor, where they demonstrate actions opposite to those of the benzodiazepines and elicit anxiogenic effects. We tested the acute cardiovascular effects of beta-carbolines in conscious unrestrained rats. Intravenous infusion of ethyl-beta-carboline-3-carboxylate (BCCE) or methyl-beta-carboline-3-carboxylate (BCCM) caused dose-related decreases in heart rate. Pretreatment with RO 15-1788 (10.0 mg/kg, i.v.), a benzodiazepine receptor antagonist, or atropine (1.0 mg/kg, i.v.) prevented the bradycardia elicited by BCCE (3.0 mg/kg, i.v.). In contrast, tetrahydro-beta-carboline (THBC; 3.0 mg/kg, i.v.) increased both heart rate and blood pressure significantly as compared with controls. However, larger doses of THBC failed to elicit further increases in heart rate or blood pressure. These experiments indicate that in conscious unrestrained rats, beta-carbolines given acutely do not routinely elicit the cardiovascular changes normally associated with stress or anxiety. We next extended our observations to rats given the beta-carboline noreleagnine, 2 mg/kg, or vehicle twice daily intraperitoneally for 4 weeks. The rats were fed either a high salt (8%) or a low salt (0.9%) diet. At 4 weeks, rats given noreleagnine and high salt had higher tail cuff pressures (146 +/- 4 vs. 134 +/- 4 mmHg) than those given noreleagnine and low salt. However, with direct arterial measurement, these differences disappeared. These data suggest that beta-carbolines do not provide a useful model for investigating the effects of chronic stress on cardiovascular function in rats.

Probing the V1a vasopressin receptor binding site with pyroglutamate-substituted linear antagonists.

We have synthesized eight analogues of the linear vasopressin antagonist DTyr(Et)2-Phe3-Gln4-Asn5-Arg6-Pro7-Arg8-Tyr(NH2)9 substituted with L-, or D-, pyroglutamate at position-1, Asn or Val at position-4 and Arg or Met at position 6. All of these peptides bound to the V1a vasopressin receptor with affinities ranging 33.6-5, 470 nM. Of this series, only two peptides, [LpGlu1Val4Arg6Tyr(NH2)9]AVP Kd = 48.4 nM and [DpGlu1Val4Arg6Tyr(NH2)9]AVP Kd = 691 nM, bound to the V2 vasopressin receptor. All of the neurohypophysial hormone receptors studied (V1a VPR, V2 VPR and OTR) were found to be stereoselective with respect to the N-terminal pGlu residue. The effect on binding characteristics of L-pGlu1 and D-pGlu1 analogues was dependent on both the sequence of the peptide and on the receptor subtype in question. From these data we found that peptide 5, which has the structure DpGlu-DTyr(Et)-Phe-Val-Asn-Arg-Pro-ARg-Tyr(NH2), exhibited the highest V1a/OTR selectivity reported to date (V1aVPR Kd = 82 nM; OTR no binding at 10 microM). As such, peptide 5 will provide useful leads to the development of ligands with enhanced V1a/OTR selectivity. The binding affinity and hydrophobicity of pyroglutamate-substituted peptides was compared with previously characterized V1a receptor antagonists which contained a range of position-1 substitutions. The hydrophobicity of both cyclic and linear antagonists was markedly increased relative to the agonists AVP and [Phe2Orn8]VT but increased hydrophobicity alone did not exclusively lead to high affinity antagonists. Data presented support the contention that in addition to a general increase in hydrophobicity/lipophilicity, position-1 influences the pharmacophore of vasopressin antagonists by providing molecular determinants for ligand/receptor interaction.

Neonatal sucking as an outcome variable: comparison of quantitative and clinical assessments.

A quantitative neonatal sucking measure and a clinical neonatal sucking measure were significantly related to one another, but only the quantitative measure was significantly related to prenatal alcohol and nicotine exposure. The clinical measure did not provide adequate precision to warrant its recommendation as an outcome variable at moderate exposure levels.

The influence of fine surface structures on the osseo-integration of implants.

The availability of inert materials like dense, pure Al2O3-ceramic or titanium allows the study of purely biomechanical influences of surface modulations or lacune on the osseo-integration of implants at different locations of the skeleton. The discovery of the "load-line-shadow" phenomenon in lacune of dental implants and the observation of the same effect in the grooves of hip sockets (Lindenhof type) indicate the general validity of the rules controlling the remodelling ability of bony tissue. Their application to the problems concerned with load transmission via surfaces which are mainly loaded by shear can contribute to achieve a well defined anchorage of implants.

Electromyography and computed tomography scan-guided fine needle aspiration biopsy of discrete extraocular muscle metastases.

Discrete or isolated metastasis to the extraocular muscles without an accompanying intraconal or extraconal mass is rare. Although it is estimated that 5% of orbital metastasis involves extraocular muscles only, to our knowledge there are no reports of discrete extraocular muscle involvement from renal cell carcinoma. With discrete extraocular muscle involvement, accurate localization within the infiltrated region of the muscle is of paramount importance in achieving a safe and positive biopsy. We describe a case with discrete extraocular muscle metastasis from a renal cell carcinoma, where a novel technique of FNAB (fine needle aspiration biopsy) of the medial rectus was undertaken, with EMG (electromyography) control and CT (computerized tomography) scan guidance.

The longstanding problem of flat feet.

The problems posed by flat feet to recruitment for the Army are assessed and the methods by which the Army has responded to the challenge over the years are reviewed.