We describe the incorporation of non-uniform sampling (NUS) compressed sensing (CS) into Oriented Sample (OS) Solid-state NMR for stationary aligned samples and Magic Angle Spinning (MAS) Solid-state NMR for unoriented ‘powder’ samples Both simulated and experimental results indicate that 25% to 33% of a full linearly sampled data set is required to reconstruct two-and three-dimensional solid-state NMR spectra with high fidelity. A modest increase in signal-to-noise ratio is accompanies the reconstruction.
Structure determination by solid-state NMR of proteins is rapidly advancing as result of recent developments of samples, experimental methods, and calculations. There are a number of different solid-state NMR approaches that utilize stationary, aligned samples or magic angle spinning of unoriented ‘powder’ samples, and depending on the sample and the experimental method can emphasize the measurement of distances or angles, ideally both, as sources of structural constraints. Multi-dimensional correlation spectroscopy of low-gamma nuclei such as 15N and 13C is an important step for making resonance assignments and measurements of angular restraints in membrane proteins. However, the efficiency of coherence transfer predominantly depends upon the strength of dipole-dipole interaction, and this can vary from site to site and between sample alignments, for example, during the mixing of 13C and 15N magnetization in stationary aligned and in magic angle spinning samples. Here, we demonstrate that the efficiency of polarization transfer can be improved by using adiabatic demagnetization and remagnetization techniques on stationary aligned samples; and proton assisted insensitive nuclei cross-polarization in magic angle sample spinning samples. Adiabatic cross-polarization technique provides an alternative mechanism for spin-diffusion experiments correlating 15N/15N and 15N/13C chemical shifts over large distances. Improved efficiency in cross-polarization with 40% – 100% sensitivity enhancements are observed in proteins and single crystals, respectively. We describe solid-state NMR experimental techniques that are optimal for membrane proteins in liquid crystalline phospholipid bilayers under physiological conditions. The techniques are illustrated with data from both single crystals of peptides and of membrane proteins in phospholipid bilayers.
membrane proteins; phospholipid bilayers; adiabatic methods; spin-exchange; spin-diffusion; separated local field spectroscopy; structure determination
Wu-Ling-San (WLS) formula has been proved to prevent calcium oxalate nephrolithiasis both in vitro and in vivo. This is the first prospective, randomized and placebo-controlled clinical trial of WLS in calcium oxalate nephrolithiasis prevention. All patients who enrolled were asked to drink enough fluid to urinate at least 2 L daily during the study period. A 24-hour urine collection was performed to establish the baseline levels of multiple urinary parameters before taking the medicine. The patients were randomized and divided into two groups. The medication group took 2 gm WLS formula three times daily for 1 month. The control group took 2 gm placebo three times daily for 1 month. A 24-hour urine collection was performed to evaluate multiple urinary and serum parameters from all patients during the study period. A total of 39 patients were enrolled and 28 patients completed the study. Fourteen patients were allocated to WLS group and 14 patients to placebo group. After treatment, the mean urine output level increased to 2796.4 ± 525.7 ml/day (percentage of change, 13.9 %) in the WLS formula group. With placebo therapy, the mean decreased slightly to 2521.4 ± 762.7ml/day (percentage of change, −5.7 %). The percentage of change was significantly different between the two groups (independent t-test, P=0.02). No patient complained of side effects, such as fatigue, dizziness, musculoskeletal symptoms, or gastrointestinal disturbance. WLS formula is a promising adjunct to surgical and medical management of kidney stones. Active therapy with WLS formula has a positive effect on diuresis without leading to electrolyte imbalance.
Calcium oxalate; Urinary Stone; Traditional Chinese Medicine; Wu-Ling-San Formula; Nephrolithiasis
Background and Purpose
Leukoaraiosis (LA) and male sex have been associated with decreased cerebrovascular reactivity, which potentially adversely affects tissue viability in acute stroke. Therefore, we aimed to elucidate the contribution of LA-severity and sex to the extent of the hyperacute ischemic core volume following intracranial large artery occlusion (ILAO).
We analyzed data from 87 patients with acute ILAO who had acute multimodal CT-imaging. LA-severity was assessed using the van Swieten scale on non-contrast CT. CT-perfusion (CTP) data were analyzed using automatic calculation of the mean transit time (MTT) and hyperacute cerebral blood volume (CBV) defects. Multivariate linear and logistic regression analyses were used to identify independent predictors of the hyperacute infarct-volume.
Severe LA (VSS 3–4; OR 43.22, 95%-CI 6.26–298.42, p<0.001) and male sex (OR 7.52, 95%-CI 1.38–40.86, p=0.020) were independently associated with a hyperacute CBV-lesion >25 mL on multivariate logistic regression analysis. Multivariate linear regression analysis confirmed the association between severe LA (p<0.001) and male sex (p=0.01) with larger CBV lesions. There was no significant difference in the absolute or relative MTT-lesion volumes when stratified by LA severity or sex. Women had significantly smaller CBV-lesion volumes compared to men (p=0.036).
Severe LA and male sex are associated with larger infarct cores, which adds to the notion that sex and LA alter the brain’s intrinsic susceptibility to acute cerebral ischemia. Future, larger studies are needed to confirm our observation that women have smaller core volumes and its significance.
Acute Stroke; CT; focal ischemia; leukoaraiosis; Women & Minorities
In this paper we describe enzyme-responsive fluorogenic micellar nanoparticles with detectable spectrophotometric properties unique to the particles and their aggregated state. These micelles are assembled from peptide-polymer amphiphiles (PPAs) labeled with either fluorescein or rhodamine. This is achieved by labelling otherwise similar block copolymer amphiphiles with each of the dyes. When mixed together, signals from the FRET-pairs can be utilized to detect particle assembly and hence enzymatic activity. Furthermore, we show FRET signals within the shell of the assembled micelles can be used to estimate particle stability (critical aggregation concentration) and enable a determination of intraparticle distances between amphiphiles in the micellar aggregates leading to elucidation of the packing arrangement of amphiphilic copolymers within the micelles.
Activation of type 1 angiotensin (AT1) receptors causes hypertension, leading
to progressive kidney injury. AT1 receptors are expressed on immune cells, and previous
studies have identified a role for immune cells in angiotensin II–dependent hypertension.
We, therefore, examined the role of AT1 receptors on immune cells in the pathogenesis of
hypertension by generating bone marrow chimeras with wild-type donors or donors lacking
AT1A receptors (BMKO). The 2 groups had virtually identical blood pressures at baseline,
suggesting that AT1 receptors on immune cells do not make a unique contribution to the
determination of baseline blood pressure. By contrast, in response to chronic angiotensin II
infusion, the BMKOs had an augmented hypertensive response, suggesting a protective effect of
AT1 receptors on immune cells with respect to blood pressure elevation. The BMKOs had
50% more albuminuria after 4 weeks of angiotensin II–dependent hypertension.
Angiotensin II–induced pathological injury to the kidney was similar in the experimental
groups. However, there was exaggerated renal expression of the macrophage chemokine monocyte
chemoattractant protein 1 in the BMKO group, leading to persistent accumulation of macrophages in
the kidney. This enhanced mononuclear cell infiltration into the BMKO kidneys was associated with
exaggerated renal expression of the vasoactive mediators interleukin-1β and
interleukin-6. Thus, in angiotensin II-induced hypertension, bone marrow-derived AT1
receptors limited mononuclear cell accumulation in the kidney and mitigated the chronic hypertensive
response, possibly through the regulation of vasoactive cytokines.
angiotensin II; hypertension; inflammation; kidney diseases; lymphocytes
Repetitive Ag encounter, coupled with dynamic changes in Ag density and inflammation, imparts phenotypic and functional heterogeneity to memory virus-specific CD8 T cells in persistently infected hosts. For herpesvirus infections, which cycle between latency and reactivation, recent studies demonstrate that virus-specific T cell memory is predominantly derived from naïve precursors recruited during acute infection. Whether functional memory T cells to viruses that persist in a non-latent, low-level infectious state (smoldering infection) originate from acute infection-recruited naïve T cells is not known. Using mouse polyomavirus (MPyV) infection, we previously showed that virus-specific CD8 T cells in persistently infected mice are stably maintained and functionally competent; however, a sizeable fraction of these memory T cells are short-lived. Further, we found that naïve anti-MPyV CD8 T cells are primed de novo during persistent infection and contribute to maintenance of the virus-specific CD8 T cell population and its phenotypic heterogeneity. Using a new MPyV-specific TCR transgenic system, we now demonstrate that virus-specific CD8 T cells recruited during persistent infection possess multi-cytokine effector function, have strong replication potential, express a phenotype profile indicative of authentic memory capability, and are stably maintained. In contrast, CD8 T cells recruited early in MPyV infection express phenotypic and functional attributes of clonal exhaustion, including attrition from the memory pool. These findings indicate that naïve virus-specific CD8 T cells recruited during persistent infection contribute to preservation of functional memory against a smoldering viral infection.
The relationship between obesity and a single nucleotide polymorphism (SNP), rs5443 (C825T), in the guanine nucleotide binding protein beta polypeptide 3 (GNB3) gene is currently inconsistent. In this study, we aimed to reassess whether the GNB3 rs5443 SNP could influence obesity and obesity-related metabolic traits in a Taiwanese population. A total of 983 Taiwanese subjects with general health examinations were genotyped. Based on the criteria defined by the Department of Health in Taiwan, the terms “overweight” and “obesity” are defined as 24 ≦ BMI < 27 and BMI ≧ 27, respectively. Compared to the carrier of the combined CT + TT genotypes of the GNB3 rs5443 polymorphism, triglyceride was significantly higher for the carrier of CC genotype in the complete sample population (128.2 ± 93.2 vs. 114.3 ± 79.1 mg/dl; P = 0.041). In addition, the carriers of CC variant had a higher total cholesterol than those with the combined CT + TT variants (194.5 ± 36.8 vs. 187.9 ± 33.0 mg/dl; P = 0.019) in the complete sample population. In the normal controls, both triglyceride (P = 0.018) and total cholesterol (P = 0.011) were also significantly higher in the CC homozygotes than in the combined CT + TT genotypes. However, the GNB3 rs5443 SNP did not exhibit any significant association with obesity or overweight among the subjects. Our study indicates that the CC genotype of the GNB3 rs5443 SNP may predict higher obesity-related metabolic traits such as triglyceride and total cholesterol in non-obese Taiwanese subjects (but not in obese subjects).
G-protein; Metabolic phenotypes; Obesity; Single nucleotide polymorphisms
Retinoic acid (RA) has been believed to be an anticancer drug for a long history. However, the molecular mechanisms of RA actions on cancer cells remain diverse. In this study, the dose-dependent inhibition of RA on DU145 cell proliferation was identified. Interestingly, RA treatment triggered p35 cleavage (p25 formation) and Cdk5 overactivation, and all could be blocked by Calpain inhibitor, Calpeptin (CP). Subsequently, RA-triggered DU145 apoptosis detected by sub-G1 phase accumulation and Annexin V staining could also be blocked by CP treatment. Furthermore, RA-triggered caspase 3 activation and following Cdk5 over-activation were destroyed by treatments of both CP and Cdk5 knockdown. In conclusion, we report a new mechanism in which RA could cause apoptosis of androgen-independent prostate cancer cells through p35 cleavage and Cdk5 over-activation. This finding may contribute to constructing a clearer image of RA function and bring RA as a valuable chemoprevention agent for prostate cancer patients.
1H-irradiation under mismatched Hartmann-Hahn conditions provides an alternative mechanism for carrying out 15N/13C transfers in triple-resonance heteronuclear correlation spectroscopy (HETCOR) on stationary samples of single crystals and aligned samples of biopolymers, which improve the efficiency especially when the direct 15N-13C dipolar couplings are small. In many cases, the sensitivity is improved by taking advantage of the 13Cα labeled sites in peptides and proteins with 13C detection. The similarities between experimental and simulated spectra demonstrate the validity of the recoupling mechanism and identify the potential for applying these experiments to virus particles or membrane proteins in phospholipid bilayers; however, further development is needed in order to derive quantitative distance and angular constraints from these measurements.
13C NMR; solid-state NMR; oriented samples; cross-polarization; triple-resonance
Human polyomaviruses are associated with substantial morbidity in immunocompromised patients, including those with HIV/AIDS, recipients of bone marrow and kidney transplants, and individuals receiving immunomodulatory agents for autoimmune and inflammatory diseases. No effective antipolyomavirus agents are currently available, and no host determinants have been identified to predict susceptibility to polyomavirus-associated diseases. Using the mouse polyomavirus (MPyV) infection model, we recently demonstrated that perforin-granzyme exocytosis, tumor necrosis factor alpha (TNF-α), and Fas did not contribute to control of infection or virus-induced tumors. Gamma interferon (IFN-γ) was recently shown to inhibit replication by human BK polyomavirus in primary cultures of renal tubular epithelial cells. In this study, we provide evidence that IFN-γ is an important component of the host defense against MPyV infection and tumorigenesis. In immortalized and primary cells, IFN-γ reduces expression of MPyV proteins and impairs viral replication. Mice deficient for the IFN-γ receptor (IFN-γR−/−) maintain higher viral loads during MPyV infection and are susceptible to MPyV-induced tumors; this increased viral load is not associated with a defective MPyV-specific CD8+ T cell response. Using an acute MPyV infection kidney transplant model, we further show that IFN-γR−/− donor kidneys harbor higher MPyV levels than donor kidneys from wild-type mice. Finally, administration of IFN-γ to persistently infected mice significantly reduces MPyV levels in multiple organs, including the kidney, a major reservoir for persistent mouse and human polyomavirus infections. These findings demonstrate that IFN-γ is an antiviral effector molecule for MPyV infection.
By incorporating homonuclear decoupling on both the 1H and 13C channels it is feasible to obtain high-resolution two-dimensional separated local field spectra of peptides and proteins that are 100% labeled with 13C. Dual-PISEMO (Polarization Inversion Spin Exchange Modulated Observation) can be performed as a conventional two-dimensional experiment, or with windowed detection as a one-dimensional experiment that offers flexibility as a building block for Shiftless and other multidimensional triple-resonance experiments with the inclusion of 15N irradiation. The triple-resonance MAGC probe used to perform these experiments at 500 MHz is described.
13C NMR; PISEMO; solid-state NMR; oriented samples
This review provides a practical overview of the excess cancer risks related to radiation from medical imaging. Primary care physicians should have a basic understanding of these risks. Because of recent attention to this issue, patients are more likely to express concerns over radiation risk. In addition, physicians can play a role in reducing radiation risk to their patients by considering these risks when making imaging referrals. This review provides a brief overview of the evidence pertaining to low-level radiation and excess cancer risks and addresses the radiation doses and risks from common medical imaging studies. Specific subsets of patients may be at greater risk from radiation exposure, and radiation risk should be considered carefully in these patients. Recent technical innovations have contributed to lowering the radiation dose from computed tomography, and the referring physician should be aware of these innovations in making imaging referrals.
To ascertain the consequences of severe leukopenia and the tempo of recovery, we studied the immunity of 56 adult patients treated for multiple sclerosis or systemic sclerosis with autologous CD34 cell transplantation using extremely lymphoablative conditioning. NK cell, monocyte, and neutrophil counts recovered to normal by 1 month; dendritic cell and B cell counts by 6 months; and T cell counts by 2 years posttransplant, although CD4 T cell counts remained borderline low. Initial peripheral expansion was robust for CD8 T cells but only moderate for CD4 T cells. Subsequent thymopoiesis was slow, especially in older patients. Importantly, levels of antibodies, including autoantibodies, did not drop substantially. Infections were frequent during the first 6 months, when all immune cells were deficient, and surprisingly rare (0.21 per patient year) at 7–24 months posttransplant, when only T cells (particularly CD4 T cells) were deficient. In conclusion, peripheral expansion of CD8 but not CD4 T cells is highly efficient. Prolonged CD4 lymphopenia is associated with relatively few infections, possibly due to antibodies produced by persisting pretransplant plasma cells.
Immunodeficiency; T lymphocytes; B lymphocytes; Autoimmunity
Areas of hypoattenuation in the liver which do not have mass effect are typically thought to represent focal fatty infiltration. Rarely, tumors can present without mass effect in the liver. We present a case in which areas of liver hypoattenuation which were initially thought to represent focal fatty infiltration on CT due to lack of mass effect had abnormal uptake on a FDG PET/CT exam; these areas were due to secondary hepatic involvement from non-Hodgkin’s lymphoma.
Liver; Lymphoma; Positron Emission Tomography; PET
Antiplatelet therapy remains one of the cornerstones in the management of non-cardioembolic ischemic stroke. However, a significant percentage of patients have concomitant gastroesophageal reflux or peptic ulcer disease that requires acid-reducing medications, the most powerful and effective being the proton pump inhibitors (PPIs). Antiplatelet efficacy, at least in vivo, and particularly for clopidogrel, has been shown to be reduced with concomitant proton pump inhibitor use. Whether this is clinically relevant is not clear from the limited studies available.
We conducted an extensive review of studies available on Medline related to pharmacodynamic interactions between the antiplatelet medications and proton pump inhibitors as well as clinical studies that addressed this potential interaction.
Based on the present pharmacodynamic and clinical studies we did not find a significant interaction that would reduce the efficacy of antiplatelet agents with concomitant user of proton pump inhibitors.
Patients on antiplatelet agents after a transient ischemic attack or ischemic stroke can safely use aspirin, and extended release dipyridamole/aspirin with proton pump inhibitors. Patients on clopidogrel may use other acid-reducing drugs besides proton pump inhibitors. In rare cases where proton pump inhibitors and clopidogrel have to be used concurrently, careful close monitoring for recurrent vascular events is required.
proton pump inhibitors; antiplatelet medications; clopidogrel; ischemic stroke; cardiovascular events
In this study, we aimed to explore whether a common single nucleotide polymorphism (SNP), rs266729 (−11,377C > G), in the adiponectin C1Q and collagen domain containing (ADIPOQ) gene could influence weight reduction and fat change under sibutramine therapy in an obese population. There were 131 obese Taiwanese patients, including 44 in the placebo group and 87 in the sibutramine (10 mg daily) group. We assessed the measures of weight loss and body fat reduction at the end of the 12-week treatment period by analysis of covariance (ANCOVA) models using gender, baseline weight, and baseline percent body fat as covariates. By comparing the placebo and sibutramine groups with ANCOVA, our data revealed a strong effect of sibutramine on percent body fat loss (1.9 ± 0.3 vs 4.6 ± 0.5%; P < 0.001) and on weight reduction (2.8 ± 2.0 vs 7.9 ± 1.6 kg; P < 0.001) for subjects with the CC genotype. On the contrary, sibutramine had no significant effect on percent body fat loss or on weight loss in the GG and GC individuals. The results suggest that the SNP rs266729 of the ADIPOQ gene may contribute to weight reduction and fat loss in response to sibutramine therapy in Taiwanese obese patients.
body fat; obesity; sibutramine; single nucleotide polymorphisms; weight loss
Chronic hepatitis C (CHC) patients often stop pursuing interferon-alfa and ribavirin (IFN-alfa/RBV) treatment because of the high cost and associated adverse effects. It is highly desirable, both clinically and economically, to establish tools to distinguish responders from nonresponders and to predict possible outcomes of the IFN-alfa/RBV treatments. Single nucleotide polymorphisms (SNPs) can be used to understand the relationship between genetic inheritance and IFN-alfa/RBV therapeutic response. The aim in this study was to establish a predictive model based on a pharmacogenomic approach. Our study population comprised Taiwanese patients with CHC who were recruited from multiple sites in Taiwan. The genotyping data was generated in the high-throughput genomics lab of Vita Genomics, Inc. With the wrapper-based feature selection approach, we employed multilayer feedforward neural network (MFNN) and logistic regression as a basis for comparisons. Our data revealed that the MFNN models were superior to the logistic regression model. The MFNN approach provides an efficient way to develop a tool for distinguishing responders from nonresponders prior to treatments. Our preliminary results demonstrated that the MFNN algorithm is effective for deriving models for pharmacogenomics studies and for providing the link from clinical factors such as SNPs to the responsiveness of IFN-alfa/RBV in clinical association studies in pharmacogenomics.
chronic hepatitis C; artificial neural networks; interferon; pharmacogenomics; ribavirin; single nucleotide polymorphisms
False-positive FDG uptake has been noted in a wide range of benign processes. In this report, we describe a case of FDG uptake in unilateral sternoclavicular synovitis which mimicked an internal mammary node in appearance. Knowledge of this potential false-positive is particularly important in breast cancer patients with a propensity of internal mammary nodal metastases.
Positron emission tomography; fluorodeoxyglucose; sternoclavicular joint; breast cancer; synovitis; arthritis
Nowadays diabetic nephropathy (DN) is the most common cause of end-stage renal disease (ESRD). Recent studies have demonstrated that the myosin, heavy chain 9, non-muscle (MYH9) gene is associated with ESRD in African Americans. In this study, we tested the hypothesis that a common single nucleotide polymorphism rs16996677 in the MYH9 gene may contribute to the etiology of DN in type 2 diabetes (T2D) in a Taiwanese population with T2D. There were 180 T2D patients diagnosed with DN and 178 age- and sex-similar T2D without DN controls. Single locus analyses showed no significant main effects of MYH9 rs16996677 on the risk of DN in T2D. The results suggest that the rs16996677 SNP in MYH9 may not contribute to the risk of DN in T2D in Taiwanese T2D patients.
diabetic nephropathy; end-stage renal disease; single nucleotide polymorphisms; type 2 diabetes
In the studies of genomics, it is essential to select a small number of genes that are more significant than the others for the association studies of disease susceptibility. In this work, our goal was to compare computational tools with and without feature selection for predicting chronic fatigue syndrome (CFS) using genetic factors such as single nucleotide polymorphisms (SNPs).
We employed the dataset that was original to the previous study by the CDC Chronic Fatigue Syndrome Research Group. To uncover relationships between CFS and SNPs, we applied three classification algorithms including naive Bayes, the support vector machine algorithm, and the C4.5 decision tree algorithm. Furthermore, we utilized feature selection methods to identify a subset of influential SNPs. One was the hybrid feature selection approach combining the chi-squared and information-gain methods. The other was the wrapper-based feature selection method.
The naive Bayes model with the wrapper-based approach performed maximally among predictive models to infer the disease susceptibility dealing with the complex relationship between CFS and SNPs.
We demonstrated that our approach is a promising method to assess the associations between CFS and SNPs.
Major depressive disorder (MDD) is one of the most common mental disorders worldwide. Single nucleotide polymorphisms (SNPs) can be used in clinical association studies to determine the contribution of genes to drug efficacy. A common SNP in the brain-derived neurotrophic factor (BDNF) gene, a methionine (Met) substitution for valine (Val) at codon 66 (Val66Met), is a candidate SNP for influencing antidepressant treatment outcome. In this study, our goal was to determine the relationship between the Val66Met polymorphism in the BDNF gene and the rapid antidepressant response to venlafaxine in a Taiwanese population with MDD. Overall, the BDNF Val66Met polymorphism was found not to be associated with short-term venlafaxine treatment outcome. However, the BDNF Val66Met polymorphism showed a trend to be associated with rapid venlafaxine treatment response in female patients. Future research with independent replication in large sample sizes is needed to confirm the role of the BDNF Val66Met polymorphism identified in this study.
antidepressant response; brain-derived neurotrophic factor; major depressive disorder; serotonin and norepinephrine reuptake inhibitor; single nucleotide polymorphisms
In the studies of genomics, it is essential to select a small number of genes that are more significant than the others for research ranging from candidate gene studies to genome-wide association studies. In this study, we proposed a Bayesian method for identifying the promising candidate genes that are significantly more influential than the others. We employed the framework of variable selection and a Gibbs sampling based technique to identify significant genes. The proposed approach was applied to a genomics study for persons with chronic fatigue syndrome. Our studies show that the proposed Bayesian methodology is effective for deriving models for genomic studies and for providing information on significant genes.
Bayesian variable selection; genomics; Gibbs sampling; variable selection
The activation and entry of antigen-specific CD8+ T cells into the central nervous system is an essential step towards clearance of West Nile virus (WNV) from infected neurons. The molecular signals responsible for the directed migration of virus-specific T cells and their cellular sources are presently unknown. Here we demonstrate that in response to WNV infection, neurons secrete the chemokine CXCL10, which recruits effector T cells via the chemokine receptor CXCR3. Neutralization or a genetic deficiency of CXCL10 leads to a decrease in CXCR3+ CD8+ T-cell trafficking, an increase in viral burden in the brain, and enhanced morbidity and mortality. These data support a new paradigm in chemokine neurobiology, as neurons are not generally considered to generate antiviral immune responses, and CXCL10 may represent a novel neuroprotective agent in response to WNV infection in the central nervous system.
We investigated a possible synergistic effect of a macrolide and β-lactams against Streptococcus pneumoniae strains with different resistance profiles. Checkerboard and time-kill assays of erythromycin combined with penicillin or cefotaxime essentially showed indifference, suggesting that these antibiotics in combinations in vitro act substantially as individuals in their activity against S. pneumoniae.