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Michael Harris

Publications and source records attributed to Michael Harris.

18 recordsLinked to original sources
PubMed2025

Pitfalls of bacterial pan-genome analysis approaches: a case study of Mycobacterium tuberculosis and two less clonal bacterial species.

SUMMARY: Pan-genome analysis is a fundamental tool for studying bacterial genome evolution; however, the variety in methods used to define and measure the pan-genome poses challenges to the interpretation and reliability of results. Using Mycobacterium tuberculosis, a clonally evolving bacterium with a small accessory genome, as a model system, we systematically evaluated sources of variability in pan-genome estimates. Our analysis revealed that differences in assembly type (short-read versus hybrid), annotation pipeline, and pan-genome software, significantly impact predictions of core and accessory genome size. Extending our analysis to two additional bacterial species, Escherichia coli and Staphylococcus aureus, we observed consistent tool-dependent biases but species-specific patterns in pan-genome variability. Our findings highlight the importance of integrating nucleotide- and protein-level analyses to improve the reliability and reproducibility of pan-genome studies across diverse bacterial populations. AVAILABILITY AND IMPLEMENTATION: Panqc is freely available under an MIT license at https://github.com/maxgmarin/panqc.

Genome, Bacterial↗
PubMed2006

A novel neutrophil elastase inhibitor prevents elastase activation and surface cleavage of the epithelial sodium channel expressed in Xenopus laevis oocytes.

The amiloride-sensitive epithelial sodium channel (ENaC) constitutes a limiting step in sodium reabsorption across distal airway epithelium and controlling mucociliary clearance. ENaC is activated by serine proteases secreted in the extracellular milieu. In cystic fibrosis lungs, high concentrations of secreted neutrophil elastase (NE) are observed. hNE could activate ENaC and contribute to further decreased mucociliary clearance. The aims of this study were (i) to test the ability of an engineered human neutrophil elastase inhibitor (EPI-hNE4) to specifically inhibit the elastase activation of ENaC-mediated amiloride-sensitive currents (I(Na)) and (ii) to examine the effect of elastase on cell surface expression of ENaC and its cleavage pattern (exogenous proteolysis). Oocytes were exposed to hNE (10-100 microg/ml) and/or trypsin (10 microg/ml) for 2-5 min in the presence or absence of EPI-hNE4 (0.7 microm). hNE activated I(Na) 3.6-fold (p < 0.001) relative to non-treated hENaC-injected oocytes. EPI-hNE4 fully inhibited hNE-activated I(Na) but had no effect on trypsin- or prostasin-activated I(Na). The co-activation of I(Na) by hNE and trypsin was not additive. Biotinylation experiments revealed that cell surface gamma ENaC (but not alpha or beta ENaC) exposed to hNE for 2 min was cleaved (as a 67-kDa fragment) and correlated with increased I(Na). The elastase-induced exogenous proteolysis pattern is distinct from the endogenous proteolysis pattern induced upon preferential assembly, suggesting a causal relationship between gamma ENaC cleavage and ENaC activation, taking place at the plasma membrane.

Animals↗
PubMed2006

An efficient and high-throughput approach for experimental validation of novel human gene predictions.

A highly automated RT-PCR-based approach has been established to validate novel human gene predictions with no prior experimental evidence of mRNA splicing (ab initio predictions). Ab initio gene predictions were selected for high-throughput validation using predicted protein classification, sequence similarity to other genomes, colocalization with an MPSS tag, or microarray expression. Initial microarray prioritization followed by RT-PCR validation was the most efficient combination, resulting in approximately 35% of the ab initio predictions being validated by RT-PCR. Of the 7252 novel genes that were prioritized and processed, 796 constituted real transcripts. In addition, high-throughput RACE successfully extended the 5' and/or 3' ends of >60% of RT-PCR-validated genes. Reevaluation of these transcripts produced 574 novel transcripts using RefSeq as a reference. RT-PCR sequencing in combination with RACE on ab initio gene predictions could be used to define the transcriptome across all species.

Algorithms↗
PubMed2005

Oxygen and glucocorticoids modulate alphaENaC mRNA translation in fetal distal lung epithelium.

Glucocorticoid hormones play an important role in fetal lung maturation. It is unknown how they interact with changes in O2 tension, which play an important role in converting the lung from a fluid-secreting to a fluid-absorbing organ at birth. Airspace fluid absorption arises from active transepithelial Na+ transport with the amiloride-sensitive epithelial Na channel (ENaC), consisting of alpha, beta, and gamma subunits, representing the rate-limiting step under nonpathologic conditions. We investigated the individual and combined effects of dexamethasone (DEX) and PO2 on alphaENaC mRNA levels, rate of alphaENaC protein synthesis, and amiloride-sensitive short-circuit current in primary cultures of rat fetal distal lung epithelial cells. DEX significantly induced alphaENaC mRNA in fetal (3%) and postnatal (21%) O2, but increases in alphaENaC protein synthesis and function occurred only when epithelia were grown under a postnatal PO2. Sucrose density gradient analyses showed that DEX treatment of cells cultured at 3% O2 decreased the association of alphaENaC mRNA with large polysomes and enhanced the association with small polysomes. Conversely, incubation of DEX-treated cells in 21% O2 restored alphaENaC mRNA association with large polysomes. No significant changes were seen in the overall polyribosome profiles or in the distribution of mRNAs encoding beta and gamma subunits of ENaC or cytokeratin 18, indicating specific modulation of alphaENaC mRNA translation. These data suggest that postnatal O2 exposure may be important for efficient translation of the alphaENaC mRNA.

Animals↗
PubMed2005

Progesterone down-regulates the open probability of the amiloride-sensitive epithelial sodium channel via a Nedd4-2-dependent mechanism.

Activation of the mitogen-activated protein (MAP) kinase cascade by progesterone in Xenopus oocytes leads to a marked down-regulation of activity of the amiloride-sensitive epithelial sodium channel (ENaC). Here we have studied the signaling pathways involved in progesterone effect on ENaC activity. We demonstrate that: (i) the truncation of the C termini of the alphabetagammaENaC subunits results in the loss of the progesterone effect on ENaC; (ii) the effect of progesterone was also suppressed by mutating conserved tyrosine residues in the Pro-X-X-Tyr (PY) motif of the C termini of the beta and gamma ENaC subunits (beta(Y618A) and gamma(Y628A)); (iii) the down-regulation of ENaC activity by progesterone was also suppressed by co-expression ENaC subunits with a catalytically inactive mutant of Nedd4-2, a ubiquitin ligase that has been previously demonstrated to decrease ENaC cell-surface expression via a ubiquitin-dependent internalization/degradation mechanism; (iv) the effect of progesterone was significantly reduced by suppression of consensus sites (beta(T613A) and gamma(T623A)) for ENaC phosphorylation by the extracellular-regulated kinase (ERK), a MAP kinase previously shown to facilitate the binding of Nedd4 ubiquitin ligases to ENaC; (v) the quantification of cell-surface-expressed ENaC subunits revealed that progesterone decreases ENaC open probability (whole cell P(o), wcP(o)) and not its cell-surface expression. Collectively, these results demonstrate that the binding of active Nedd4-2 to ENaC is a crucial step in the mechanism of ENaC inhibition by progesterone. Upon activation of ERK, the effect of Nedd4-2 on ENaC open probability can become more important than its effect on ENaC cell-surface expression.

Aldosterone↗
PubMed2005

Observed environmental features and the physical activity of adolescent males.

BACKGROUND: It has recently been reported that adult physical activity was associated with environmental features. The aim of this study was to determine whether environmental features were associated with physical activity among male adolescents. METHODS: Physical activity levels of 210 Boy Scouts were assessed for 3 days by accelerometry during 2003 and 2004. Mean minutes of sedentary, light, and moderate-to-vigorous intensity activity were calculated. Environmental features within a 400-meter radius of each participant's home address were assessed by direct observation using the Systematic Pedestrian and Cycling Environmental Scan (SPACES) instrument. Principal component factor analysis reduced the 35 SPACES items to four factors. Hierarchical and spatial regressions were conducted with physical activity as the dependent variable and environmental factors, age, body mass index, and ethnicity as independent variables. RESULTS: Four factors were obtained: walking/cycling ease, tidiness, sidewalk characteristics, and street access and condition. Sidewalk characteristics were negatively associated with minutes of sedentary behavior while age was positively associated. Sidewalk characteristics were positively associated with minutes of light-intensity physical activity and age negatively associated. No environmental factor correlated with moderate-to-vigorous physical activity. CONCLUSIONS: A sidewalk characteristics factor, composed of sidewalk location, sidewalk material, presence of streetlights, and number and height of trees, was positively associated with light-intensity physical activity among male adolescents.

Adolescent↗
PubMed2005

Total hip arthroplasty with the APR stem and cup follow-up of a previous report.

The 2- to 5-year results of the APR-II total hip arthroplasty system (APR-II, Zimmer, Warsaw, Ind) were previously reported for 107 consecutive total hip arthroplasties performed between January 1994 and January 1995. The purpose of this study was to report the 10-year survival data in this same cohort of patients. Seventy-two (73%) of the 99 hips originally studied met the inclusion criteria for this report. None of the femoral or acetabular components had radiographic or clinical loosening. Three hips had had revision surgery for the acetabulum with 2 for recurrent dislocation and 1 for liner disassociation. All femoral components were retained. The 10-year Kaplan-Meier survivorship analysis, with failure defined as mechanical loosening, was 100% for both the cup and stem. These hip replacements have not shown any postoperative deterioration of clinical result, fixation, or wear up to 10 years.

Adult↗
PubMed2005

Viral immune evasion molecules attack the ER peptide-loading complex and exploit ER-associated degradation pathways.

The CD8+ cytotoxic-T-cell response is a potent mechanism that controls intracellular pathogens, including many viruses. To facilitate transmission, viruses often counter this response by inhibiting the cell surface display of virus-derived peptides on MHC class I molecules. More specifically, recent studies have demonstrated that viruses have evolved remarkable mechanisms to inhibit MHC class I expression by interfering with the function of the MHC class I assembly machinery (the peptide-loading complex) in the endoplasmic reticulum and/or by exploiting endoplasmic-reticulum-associated degradation pathways. These viral molecules are proving invaluable as research tools to illuminate the novel features of physiological pathways that are central to normal cell biology. Furthermore, the detailed characterization of such pathways has yielded significant new insights into host-pathogen interplay.

Animals↗
PubMed2005

Total femur replacement procedures in tumor treatment.

UNLABELLED: Replacement of bone segments after resection of a tumor-containing bone has a long history, and currently metallic implants and allografts have reasonable rates of success in terms of patient survival and restoration of useful function. Total femoral resections for extensive tumors are uncommon but clearly represent a major problem, which in the past has required hip disarticulations. In recent years, resection and replacement using custom metallic implants and more recently modular devices have allowed the patients to be restored to reasonable function. Allografts have not been used commonly for this purpose. During the past 23 years, we have done 15 total femoral replacements for osteosarcoma, chondrosarcoma, metastatic carcinoma, and an osteonecrotic fractured femur in a patient with rheumatoid arthritis and for a patient with severe deformity based on Paget's disease. Ten of these patients had allografts implanted with total hip replacement and total knee replacement implants and five patients had metallic implants. Eight of the patients died from disease, but the remaining seven have done reasonably well in terms of function and absence of complications such as fracture or infection. None of the patients required an amputation. The procedure is complex and metallic implant and allograft implantation require wide surgery and special systems of reconstruction. LEVEL OF EVIDENCE: Therapeutic study, Level IV-1 (case series). See the Guidelines for Authors for a complete description of levels of evidence.

Adolescent↗
PubMed2005

Gene and alternative splicing annotation with AIR.

Designing effective and accurate tools for identifying the functional and structural elements in a genome remains at the frontier of genome annotation owing to incompleteness and inaccuracy of the data, limitations in the computational models, and shifting paradigms in genomics, such as alternative splicing. We present a methodology for the automated annotation of genes and their alternatively spliced mRNA transcripts based on existing cDNA and protein sequence evidence from the same species or projected from a related species using syntenic mapping information. At the core of the method is the splice graph, a compact representation of a gene, its exons, introns, and alternatively spliced isoforms. The putative transcripts are enumerated from the graph and assigned confidence scores based on the strength of sequence evidence, and a subset of the high-scoring candidates are selected and promoted into the annotation. The method is highly selective, eliminating the unlikely candidates while retaining 98% of the high-quality mRNA evidence in well-formed transcripts, and produces annotation that is measurably more accurate than some evidence-based gene sets. The process is fast, accurate, and fully automated, and combines the traditionally distinct gene annotation and alternative splicing detection processes in a comprehensive and systematic way, thus considerably aiding in the ensuing manual curation efforts.

Alternative Splicing↗
PubMed2004

Contact lens fitting over intrastromal corneal rings in a keratoconic patient.

BACKGROUND: Intrastromal corneal ring segments have been used as a refractive surgery method for treatment of myopia. More recently, attempts have been made to use this surgical procedure in patients with keratoconus. CASE REPORT: A 51-year-old man with keratoconus came to the clinic for a contact lens refit. He had undergone surgical insertion of intrastromal corneal rings in his right eye 7 months earlier in an attempt to obtain acceptable vision without contact lenses. At the time of presentation, the patient was not satisfied with his vision in the right eye and requested an attempt to improve his vision with a contact lens. The resulting corneal shape after surgery presented some complications, but the patient was able to achieve 20/25-vision and adequate comfort in his right eye with a piggyback contact lens system. CONCLUSIONS: Insertion of intrastromal corneal rings may be beneficial in patients with early keratoconus or who are contact lens-intolerant. However, patients with more-advanced keratoconus are not likely to achieve adequate vision after the surgery and will require a contact lens. In addition, changes in the corneal anatomy created by the intrastromal rings may make contact lens fitting more challenging. The corneal irregularity may be exacerbated due to drastic variations in corneal elevation over the intrastromal ring and directly adjacent to the ring. This irregularity in elevation leads to difficulty in centering the lens on the eye and problems with bubbles forming under the contact lens over areas of corneal depression. When successfully fitted, contact lenses over intrastromal corneal rings can improve vision significantly.

Contact Lenses↗
PubMed2003

Endothelial cells preparing to die by apoptosis initiate a program of transcriptome and glycome regulation.

The protein-based changes that underlie the cell biology of apoptosis have been extensively studied. In contrast, mRNA- and polysaccharide-based changes have received relatively little attention. We have combined transcriptome and glycome analyses to show that apoptotic endothelial cell cultures undergo programmed changes to RNA transcript abundance and cell surface polysaccharide profiles. Although a few of the transcriptome changes were protective, most appeared to prepare cells for apoptosis by decreasing the reception and transduction of pro-survival signals, increasing pro-death signals, increasing abundance of apoptotic machinery, inhibiting cellular proliferation, recruiting phagocytes to regions of cell death, and promoting phagocytosis. Additional transcriptomal changes appeared to alter the synthesis and modification of cell surface glycosaminoglycans. The resultant reduced abundance of sulphated cell surface glycosaminoglycans may further promote cell death by inhibiting the presentation of extracellular matrix-tethered survival factors to their receptors on dying cells. We propose that the transcriptome and glycome regulation presented here synergize with previously described protein-based changes to guide the apoptotic program.

Apoptosis↗
PubMed2003

Remote photoacoustic detection of liquid contamination of a surface.

A method for the remote detection and identification of liquid chemicals at ranges of tens of meters is presented. The technique uses pulsed indirect photoacoustic spectroscopy in the 10-microm wavelength region. Enhanced sensitivity is brought about by three main system developments: (1) increased laser-pulse energy (150 microJ/pulse), leading to increased strength of the generated photoacoustic signal; (2) increased microphone sensitivity and improved directionality by the use of a 60-cm-diameter parabolic dish; and (3) signal processing that allows improved discrimination of the signal from noise levels through prior knowledge of the pulse shape and pulse-repetition frequency. The practical aspects of applying the technique in a field environment are briefly examined, and possible applications of this technique are discussed.

Journal Article↗
PubMed2003

Lidar frequency modulation vibrometry in the presence of speckle.

We report laboratory target vibration measurements that use an easily aligned and adjusted fiber-based 1.5-microm heterodyne lidar. The targets are simple spherically curved retroreflectors with well-controlled vibration frequencies and amplitudes. A rotating ground-glass screen creates Gaussian speckle. We wish to understand the modulated and fast-fading lidar returns seen from real target. We frequency demodulated the recorded laboratory data by phase differencing to provide estimates of dphi/dt, where phi is the phase of the received carrier-plus-noise phasor. Experimental results for signal strength and signal-to-noise ratio, for specific target modulation parameters, agree well with our recently developed dphi/dt correlation-function theory.

Journal Article↗
PubMed2003

Mupirocin resistance in clinical isolates of Staphylococcus aureus.

One hundred isolates of Staphylococcs aureus were collected in a laboratory serving several hospitals and clinics in southeastern Wisconsin and tested for mupirocin susceptibility. Only two isolates of S. aureus showed mupirocin resistance. The mupirocin-resistant isolates were from hospitalized patients with positive blood cultures.

Anti-Bacterial Agents↗
PubMed2002

Analysis of the performance of a coherent pulsed fiber lidar for aerosol backscatter applications.

The antenna and the Doppler estimation characteristics of a coherent pulsed lidar intended for short-range aerosol backscatter applications have been analyzed. The system used fiber-optic interconnects and operated at a wavelength of 1.548 microm. The range dependence of the signal for various bistatic and monostatic antenna configurations has been determined. The system operated in a low-pulse-energy, high-pulse-repetition-rate mode, and the Doppler estimates from the return signal were achieved with a multipulse accumulation procedure. The expected performance of the accumulation in this low-photocount regime was compared with the data obtained from the system, and a reasonable level of agreement was demonstrated.

Journal Article↗
PubMed2002

Heterodyne measurements of laser light scattering by a turbulent phase screen.

We report experiments in which a fiber-coupled heterodyne laser system operating at a wavelength of 1.5 microm is used to measure the phase fluctuations induced on a laser beam by passage through a thin layer of turbulent air and subsequent propagation through free space. We investigate the statistical properties and power spectra of the phase and its rate of change, in addition to the intensity statistics. We find that the power spectrum of the rate of change of phase has a simple negative exponential form. We discuss our results in the context of the problem of detection of phase variations over an extended turbulent atmospheric path.

Journal Article↗