Toward Standards in Clinical Microbiota Studies: Comparison of Three DNA Extraction Methods and Two Bioinformatic Pipelines.

When studying the microbiome using next-generation sequencing, the DNA extraction method, sequencing procedures, and bioinformatic processing are crucial to obtain reliable data. Method choice has been demonstrated to strongly affect the final biological interpretation. We assessed the performance of three DNA extraction methods and two bioinformatic pipelines for bacterial microbiota profiling through 16S rRNA gene amplicon sequencing, using positive and negative controls for DNA extraction and sequencing and eight different types of high- or low-biomass samples. Performance was evaluated based on quality control passing, DNA yield, richness, diversity, and compositional profiles. All DNA extraction methods retrieved the theoretical relative bacterial abundance with a maximum 3-fold change, although differences were seen between methods, and library preparation and sequencing induced little variation. Bioinformatic pipelines showed different results for observed richness, but diversity and compositional profiles were comparable. DNA extraction methods were successful for feces and oral swabs, and variation induced by DNA extraction methods was lower than intersubject (biological) variation. For low-biomass samples, a mixture of genera present in negative controls and sample-specific genera, possibly representing biological signal, were observed. We conclude that the tested bioinformatic pipelines perform equally, with pipeline-specific advantages and disadvantages. Two out of three extraction methods performed equally well, while one method was less accurate regarding retrieval of compositional profiles. Lastly, we again demonstrate the importance of including negative controls when analyzing low-bacterial-biomass samples.IMPORTANCE Method choice throughout the workflow of a microbiome study, from sample collection to DNA extraction and sequencing procedures, can greatly affect results. This study evaluated three different DNA extraction methods and two bioinformatic pipelines by including positive and negative controls and various biological specimens. By identifying an optimal combination of DNA extraction method and bioinformatic pipeline use, we hope to contribute to increased methodological consistency in microbiota studies. Our methods were applied not only to commonly studied samples for microbiota analysis, e.g., feces, but also to more rarely studied, low-biomass samples. Microbiota composition profiles of low-biomass samples (e.g., urine and tumor biopsy specimens) were not always distinguishable from negative controls, or showed partial overlap, confirming the importance of including negative controls in microbiota studies, especially when low bacterial biomass is expected.

Gut Microbiota and Colonization Resistance against Bacterial Enteric Infection.

The gut microbiome is critical in providing resistance against colonization by exogenous microorganisms. The mechanisms via which the gut microbiota provide colonization resistance (CR) have not been fully elucidated, but they include secretion of antimicrobial products, nutrient competition, support of gut barrier integrity, and bacteriophage deployment. However, bacterial enteric infections are an important cause of disease globally, indicating that microbiota-mediated CR can be disturbed and become ineffective. Changes in microbiota composition, and potential subsequent disruption of CR, can be caused by various drugs, such as antibiotics, proton pump inhibitors, antidiabetics, and antipsychotics, thereby providing opportunities for exogenous pathogens to colonize the gut and ultimately cause infection. In addition, the most prevalent bacterial enteropathogens, including Clostridioides difficile, Salmonella enterica serovar Typhimurium, enterohemorrhagic Escherichia coli, Shigella flexneri, Campylobacter jejuni, Vibrio cholerae, Yersinia enterocolitica, and Listeria monocytogenes, can employ a wide array of mechanisms to overcome colonization resistance. This review aims to summarize current knowledge on how the gut microbiota can mediate colonization resistance against bacterial enteric infection and on how bacterial enteropathogens can overcome this resistance.

Identification and validation of two peptide markers for the recognition of Clostridioides difficile MLST-1 and MLST-11 by MALDI-MS.

OBJECTIVES: Clostridioides difficile infection (CDI) has become the main cause of nosocomial infective diarrhoea. To survey and control the spread of different C. difficile strains, there is a need for suitable rapid tests. The aim of this study was to identify peptide/protein markers for the rapid recognition of C. difficile strains by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). METHODS: We analysed 44 well-characterized strains, belonging to eight different multi-locus sequence types (MLST), using ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR) MS. The amino acid sequence of two peptide markers specific for MLST-1 and MLST-11 strains was elucidated by MALDI-TOF-MS/MS. The investigation of 2689 C. difficile genomes allowed the determination of the sensitivity and specificity of these markers. C18-solid-phased extraction was used to enrich the MLST-1 marker. RESULTS: Two peptide markers (m/z 4927.81 and m/z 5001.84) were identified and characterized for MLST-1 and MLST-11 strains, respectively. The MLST-1 marker was found in 786 genomes of which three did not belong to MLST-1. The MLST-11 marker was found in 319 genomes, of which 14 did not belong to MLST-11. Importantly, all MLST-1 and MLST-11 genomes were positive for their respective marker. Furthermore, a peptide marker (m/z 5015.86) specific for MLST-15 was found in 59 genomes. We translated our findings into a fast and simple method that allowed the unambiguous identification of the MLST-1 marker on a MALDI-TOF-MS platform. CONCLUSIONS: MALDI-FTICR MS-based peptide profiling resulted in the identification of peptide markers for C. difficile MLST-1 and MLST-11.

Anharmonic Molecular Mechanics: Ab Initio Based Morse Parametrizations for the Popular MM3 Force Field.

Methodologies for creating reactive potential energy surfaces from molecular mechanics force-fields are becoming increasingly popular. To date, molecular mechanics force-fields in biochemistry and small molecule organic chemistry tend to use harmonic expressions to treat bonding stretches, which is a poor approximation in reactive and nonequilibirum molecular dynamics simulations since bonds are often displaced significantly from their equilibrium positions. For such applications there is need for a better treatment of anharmonicity. In this contribution, Morse bonding potentials have been extensively parametrized for the atom types in the MM3 force field of Allinger and co-workers using high level CCSD(T)(F12*) energies. To our knowledge this is among the first instances of a comprehensive parametrization of Morse potentials in a popular organic chemistry force field. In the context of molecular dynamics simulations, these data will: (1) facilitate the fitting of reactive potential energy surfaces using empirical valence bond approaches and (2) enable more accurate treatments of energy transfer.

Stereodynamics in NO(X) + Ar inelastic collisions.

The effect of orientation of the NO(X) bond axis prior to rotationally inelastic collisions with Ar has been investigated experimentally and theoretically. A modification to conventional velocity-map imaging ion optics is described, which allows the orientation of hexapole state-selected NO(X) using a static electric field, followed by velocity map imaging of the resonantly ionized scattered products. Bond orientation resolved differential cross sections are measured experimentally for a series of spin-orbit conserving transitions and compared with quantum mechanical calculations. The agreement between experimental results and those from quantum mechanical calculations is generally good. Parity pairs, which have previously been observed in collisions of unpolarized NO with various rare gases, are not observed due to the coherent superposition of the two j = 1/2, Ω = 1/2 Λ-doublet levels in the orienting field. The normalized difference differential cross sections are found to depend predominantly on the final rotational state, and are not very sensitive to the final Λ-doublet level. The differential steric effect has also been investigated theoretically, by means of quantum mechanical and classical calculations. Classically, the differential steric effect can be understood by considering the steric requirement for different types of trajectories that contribute to different regions of the differential cross section. However, classical effects cannot account quantitatively for the differential steric asymmetry observed in NO(X) + Ar collisions, which reflects quantum interference from scattering at either end of the molecule. This quantum interference effect is dominated by the repulsive region of the potential.

Rotational Orientation Effects in NO(X) + Ar Inelastic Collisions.

Rotational angular momentum orientation effects in the rotationally inelastic collisions of NO(X) with Ar have been investigated both experimentally and theoretically at a collision energy of 530 cm(-1). The collision-induced orientation has been determined experimentally using a hexapole electric field to select the ϵ = -1 Λ-doublet level of the NO(X) j = 1/2 initial state. Fully quantum state resolved polarization-dependent differential cross sections were recorded experimentally using a crossed molecular beam apparatus coupled with a (1 + 1') resonance-enhanced multiphoton ionization detection scheme and subsequent velocity-map imaging. To determine the NO sense of rotation, the probe radiation was circularly polarized. Experimental orientation polarization-dependent differential cross sections are compared with those obtained from quantum mechanical scattering calculations and are found to be in good agreement. The origin of the collision-induced orientation has been investigated by means of close-coupled quantum mechanical, quantum mechanical hard shell, quasi-classical trajectory (QCT), and classical hard shell calculations at the same collision energy. Although there is evidence for the operation of limiting classical mechanisms, the rotational orientation cannot be accounted for by QCT calculations and is found to be strongly influenced by quantum mechanical effects.

Steric effects and quantum interference in the inelastic scattering of NO(X) + Ar.

Rotationally inelastic collisions of NO(X) with Ar are investigated in unprecedented detail using state-to-state, crossed molecular beam experiments. The NO(X) molecules are selected in the Ω = 0.5, j = 0.5, f state and then oriented such that either the 'N' or 'O' end of the molecule is directed towards the incoming Ar atom. Velocity map ion imaging is then used to probe the scattered NO molecules in well-defined quantum states. We show that the fully quantum state-resolved differential steric asymmetry, which quantifies how the relative efficiency for scattering off the 'O' and the 'N' ends of the molecule varies with scattering angle, is strongly affected by quantum interference. Significant changes in both integral and differential cross sections are found depending on whether collisions occur with the N or O ends of the molecule. The results are well accounted for by rigorous quantum mechanical calculations, in contrast to both classical trajectory calculations and more simplistic models that provide, at best, an incomplete picture of the dynamics.

Fully quantum state-resolved inelastic scattering of NO(X) + Kr: differential cross sections and product rotational alignment.

Fully quantum state selected and resolved inelastic scattering of NO(X) by krypton has been investigated. Initial Λ-doublet state selection is achieved using an inhomogeneous hexapole electric field. Differential cross sections and even-moment polarization dependent differential cross sections have been obtained at a collision energy of 514 cm(-1) for both spin-orbit and parity conserving and changing collisions. Experimental results are compared with those obtained from quantum scattering calculations and are shown to be in very good agreement. Hard shell quantum scattering calculations are also performed to determine the effects of the different parts of the potential on the scattering dynamics. Comparisons are also made with the NO(X) + Ar system.

Anal acoustic reflectometry predicts the outcome of percutaneous nerve evaluation for faecal incontinence.

BACKGROUND: Sacral nerve stimulation (SNS) is effective for some patients with faecal incontinence. Before insertion of a costly implant, percutaneous nerve evaluation (PNE) is undertaken to identify patients likely to report success from SNS. The aim of this study was to determine whether variables of anal sphincter function measured by anal acoustic reflectometry (AAR) could predict the outcome of PNE for faecal incontinence. METHODS: Women with faecal incontinence undergoing PNE were recruited. AAR, followed by anal manometry, was performed on the day of surgery, immediately before PNE. The outcome of PNE was determined by bowel diary results and incontinence severity score. Patients with a successful PNE outcome were compared with those with an unsuccessful outcome; logistic regression analysis was used to identify any independent predictors of success. RESULTS: Fifty-two patients were recruited, of whom 32 (62 per cent) had a successful PNE outcome and 20 (38 per cent) an unsuccessful outcome. The AAR variable opening pressure was significantly greater in patients who subsequently had a successful PNE result compared with the pressure in patients who did not (28 versus 17 cmH2 O; P = 0·008). No difference was seen in the manometric equivalent, maximum resting pressure. Opening pressure was an independent predictor of success with an odds ratio of 1·08 (95 per cent confidence interval 1·01 to 1·16; P = 0·018). CONCLUSION: AAR is a sensitive test of sphincter function and can identify differences between patients who respond to PNE and those who do not. Opening pressure is an independent predictor of success in PNE, and may be of value in the selection of patients for this expensive treatment option.

Origin of collision-induced molecular orientation.

Collision-induced rotational angular momentum orientation is a fundamental property of molecular scattering, which is sensitive to the balance between attractive and repulsive forces at play during collision. Here, we quantify a new mechanism leading to orientation, which is purely quantum mechanical in origin. Although the new mechanism is quite general, and will operate more widely in atomic and molecular scattering, it is observed here for impulsive hard shell collisions, for which the orientation vanishes classically. The quantum mechanism can thus be studied in isolation from other processes. The orientation is proposed to originate from the nonlocal nature of the quantum mechanical collision encounter.

Rotational alignment effects in NO(X) + Ar inelastic collisions: a theoretical study.

Rotational angular momentum alignment effects in the rotational inelastic scattering of NO(X) with Ar have been investigated by means of close-coupled quantum mechanical, quasi-classical trajectory, and Monte Carlo hard shell scattering calculations. It has been shown that the hard shell nature of the interaction potential at a collision energy of Ecoll = 66 meV is primarily responsible for the rotational alignment of the NO(X) molecule after collision. By contrast, the alternating trend in the quantum mechanical parity resolved alignment parameters with change in rotational state Δj reflects differences in the differential cross sections for NO(X) parity conserving and changing collisions, rather than an underlying difference in the collision induced rotational alignment. This suggests that the rotational alignment and the differential cross sections are sensitive to rather different aspects of the scattering dynamics. The applicability of the kinematic apse model has also been tested and found to be in excellent agreement with exact quantum mechanical scattering theory provided the collision energy is in reasonable excess of the well depth of the NO(X)-Ar potential energy surface.

Rotational alignment effects in NO(X) + Ar inelastic collisions: an experimental study.

Rotational angular momentum alignment effects in the rotationally inelastic collisions of NO(X) with Ar have been investigated at a collision energy of 66 meV by means of hexapole electric field initial state selection coupled with velocity-map ion imaging final state detection. The fully quantum state resolved second rank renormalized polarization dependent differential cross sections determined experimentally are reported for a selection of spin-orbit conserving and changing transitions for the first time. The results are compared with the findings of previous theoretical investigations, and in particular with the results of exact quantum mechanical scattering calculations. The agreement between experiment and theory is generally found to be good throughout the entire scattering angle range. The results reveal that the hard shell nature of the interaction potential is predominantly responsible for the rotational alignment of the NO(X) upon collision with Ar.

Comparative study of anal acoustic reflectometry and anal manometry in the assessment of faecal incontinence.

BACKGROUND: Anal acoustic reflectometry (AAR) is a reproducible technique providing a novel physiological assessment of anal sphincter function. It may have advantages over conventional anal manometry. The aims of this study were to determine the ability of AAR and anal manometry to identify changes in anal sphincter function in patients with faecal incontinence (FI) and to relate these changes to the severity of FI. METHODS: Women with FI underwent assessment with AAR and anal manometry. All patients completed the Vaizey FI questionnaire and were classified according to symptom type (urge, passive or mixed) and integrity of the anal sphincters. The ability of AAR and anal manometry to correlate with symptom severity was evaluated. AAR was compared with anal manometry in detecting differences in anal sphincter function between symptomatic subgroups, and patients with and without a sphincter defect. RESULTS: One hundred women with FI were included in the study. The AAR variables opening pressure, opening elastance, closing elastance and squeeze opening pressure correlated with symptom severity, whereas the manometric measurements maximum resting pressure and maximum squeeze pressure did not. Unlike anal manometry, AAR was able to detect differences in anal sphincter function between different symptomatic subgroups. [corrected]. An anal sphincter defect was not associated with a significant change in anal sphincter function determined by either AAR or anal manometry. CONCLUSION: In the assessment of women with FI, AAR variables correlated with symptom severity and could distinguish between different symptomatic subgroups. AAR may help to guide management in these patients.

Fully Λ-doublet resolved state-to-state differential cross-sections for the inelastic scattering of NO(X) with Ar.

Fully Λ-doublet resolved state-to-state differential cross-sections (DCSs) for the collisions of the open-shell NO(X, (2)Π(1/2), ν = 0, j = 0.5) molecule with Ar at a collision energy of 530 cm(-1) are presented. Initial state selection of NO(X, (2)Π(1/2), j = 0.5, f) was performed using a hexapole so that the (low field seeking) parity of ε = -1, corresponding to the f component of the Λ-doublet, could be selected uniquely. Although the Λ-doublet levels lie very close in energy to one another and differ only in their relative parities, they exhibit strikingly different DCSs. Both spin-orbit conserving and spin-orbit changing collisions have been studied, and the previously unobserved structures in the fully quantum state-to-state resolved DCSs are shown to depend sensitively on the change in parity of the wavefunction of the NO molecule on collision. In all cases, the experimental data are shown to be in excellent agreement with rigorous quantum mechanical scattering calculations.

Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line.

The primary objective of this study was to determine the sequence of biochemical signaling events that occur after modulation of the cellular redox state in the B cell lymphoma line, PW, with emphasis on the role of mitochondrial signaling. L-Buthionine sulphoximine (BSO), which inhibits gamma glutamyl cysteine synthetase (gammaGCS), was used to modulate the cellular redox status. The sequence and role of mitochondrial events and downstream apoptotic signals and mediators was studied. After BSO treatment, there was an early decline in cellular glutathione (GSH), followed by an increase in reactive oxygen species (ROS) production, which induced a variety of apoptotic signals (detectable at different time points) in the absence of any external apoptotic stimuli. The sequence of biochemical events accompanying apoptosis included a 95% decrease in total GSH and a partial (25%) preservation of mitochondrial GSH, without a significant increase in ROS production at 24h. Early activation and nuclear translocation of the nuclear factor kappa B subunit Rel A was observed at approximately 3h after BSO treatment. Cytochrome c release into the cytosol was also seen after 24h of BSO treatment. p53 protein expression was unchanged after redox modulation for up to 72 h, and p21waf1 independent loss of cellular proliferation was observed. Surprisingly, a truncated form of p53 was expressed in a time-dependent manner, beginning at 24h after BSO incubation. Irreversible commitment to apoptosis occurred between 48 and 72 h after BSO treatment when mitochondrial GSH was depleted, and there was an increase in ROS production. Procaspase 3 protein levels showed a time-dependent reduction following incubation with BSO, notably after 48 h, that corresponded with increasing ROS levels. At 96 h, caspase 3 cleavage products were detectable. The pan-caspase inhibitor zVADfmk, partially blocked the induction of apoptosis at 48 h, and was ineffective after 72 h. PW cells could be rescued from apoptosis by removing them from BSO after up to 48, but not 72 h incubation with BSO. Mitochondrial transmembrane potential (DeltaPsi(m)) remained intact in most of the cells during the 72 h observation period, indicating that DeltaPsi(m) dissipation is not an early signal for the induction of redox dependent apoptosis in PW cells. These data suggest that a decrease in GSH alone can act as a potent early activator of apoptotic signaling. Increased ROS production following mitochondrial GSH depletion, represents a crucial event, which irreversibly commits PW cells to apoptosis.

Rotenone-induced G2/M cell cycle arrest and apoptosis in a human B lymphoma cell line PW.

Concentrations of rotenone (ROT) that block electron flow through mitochondrial complex I (100 nM) did not significantly alter either cell viability or the growth of PW cells. However, 10- to 50-fold higher concentrations (1-5 microM) were found to induce a dose-dependent cell cycle arrest predominantly at the G2/M stage of the cycle and apoptosis. Apoptosis was dependent on the cell cycle arrest, since apoptosis but not the G2/M arrest was prevented with the broad spectrum caspase inhibitor zVADfmk. Biochemical features of apoptosis included mitochondrial cytochrome c release, reactive oxygen species generation, and the activation of procaspase 3. Thus, ROT inhibition of mitochondrial electron transport may be insufficient to induce apoptosis in PW cells. Instead, apoptosis in these cells occurs as a consequence of disruption of the cell cycle and is only indirectly dependent upon mitochondrial electron transport.

Long-term outcome of hairy cell leukemia treated with 2-chlorodeoxyadenosine.

The long-term results of both pretreated and previously untreated patients with hairy cell leukemia (HCL) using uniformly a single 7-day course of 2-chlorodeoxyadenosine (2-CdA) by continuous infusion are reported. In addition, the probability of obtaining another response with this drug in patients who relapsed after 2-CdA treatment will be addressed. Forty-two consecutive patients (32 men, 10 women) with a median age of 56 years (range 32-75) at the time of initiation of 2-CdA treatment were analyzed. Ten patients were pretreated with either splenectomy (n=6) or interferon a (n=8) or deoxycoformycin (dCF) (n=3) or with all procedures in sequence. Two patients who did not respond to dCF did respond to 2-CdA. Median time to start of 2-CdA treatment of the ten pretreated patients was 47 months (10-160); 41 of the 42 (98%) achieved CR, and one patient reached a good partial response with a single cycle of 2-CdA. Ten of the 42 patients had no toxicities at all. Toxicities (WHO grades I-IV) were mainly of grades I and II; in one patient with a preexisting brain injury grade III neurotoxicity was seen, and one patient suffered a grade-IV infectious complication. Bone marrow biopsies were performed at the time of recovery of hematopoiesis, thereafter at 2- to 3-month intervals, then at 6 months, and finally annually in all 42 patients. Median follow-up is 32 months (2-72). Disease-free survival from start of 2-CdA treatment is 75% at 6 years; 6/42 patients relapsed. Three of these patients were treated with 2-CdA again. All three patients reached another CR (+1, +2, +13). Four of the 42 patients had a second malignancy (carcinomas of the bladder, breast, cervix, prostate gland) before receiving 2-CdA. One patient died in CR due to the second malignancy. 2-CdA is a safe and effective treatment of HCL, inducing complete remissions in the majority of patients with only a single cycle of 2-CdA and a paucity of toxicities. Responses are durable and long lasting. Patients relapsing following a treatment with 2-CdA seem to respond to this drug again.

[Ostrich farming from the perspective of veterinarians]. / Straussenhaltung aus der Sicht des Tierarztes.

The present paper is an attempt to relate the etiology of 54 cases with diseased/deceased ostriches to criterions of keeping, feeding, climate and infectious diseases. Despite the fact that some of the damages diagnosed seem to be avoidable the criterions of the law for the prevention of cruelty to animals are not to be fulfilled since the adverse climatic circumstances in Germany.