Integrated study of group B streptococcus and human ureaplasmas : the paradigm shifts

Group B streptococcus (GBS, S. agalactiae) and human ureaplasmas (U. parvumand U. urealyticum) are two clinically and phylogenetically related, potentialperinatal pathogens. Their relationships between genotypes and pathogenesis ofGBS and ureaplasma infection were still not well understood, one of the reason isthat both of them are still short of a very practical genotyping system. In the study,to solve the above problem we developed genotyping systems for the organisms (thesecond section). For human ureaplasmas, based on four genes/gene clusters (rRNAgene clusters, the elongation factor Tu genes, urease gene complexes and multiplebanded antigen genes), we designed many primer pairs suitable for developing species identification assays for the two newly established human ureaplasmaspecies (U. parvum and U. urealyticum). Further, based on the heterogeneity ofureaplasma multiple banded antigen gene (which contains species- and serovar-specific regions), we developed genotyping methods for each ureaplasma species.For GBS, based on three sets of molecular markers (capsular polysaccharidesynthesis gene clusters, surface protein antigen genes and mobile genetic elements),we developed a genotyping system. The primary evaluation of the genotypingsystems showed that the genotyping systems were practical alternative assays forthe conventional serotyping and they will be useful to further explore therelationships between genotypes and pathogenesis of GBS and ureaplasmainfection. In the study, we introduced novel data and tools into GBS and ureaplasmastudies especially from genomic- and bioinformatics-based molecular microbiology(the third section). For two newly established human ureaplasma species, based onthe U. parvum serovar-3 genome, and using the above four important genes/geneclusters, we exposed some interesting problems in the understanding of newureaplasma taxonomy especially in the post genomic era. For GBS, we studied thetwo published full genomes and exposed some new problems or possible future newresearch fields. In particular we found the two finished and one ongoing GBSgenomes were all non-typical and suggest that future genomic project had better have genetic population structure viewpoint. Finally, we suggested that integratedstudies of the two potential or conditional perinatal pathogens, from the viewpointof evolution, would provide a new understanding angle of the pathogenesis of thetwo organisms. Studies suggested that during coevolution, human ureaplasmas(especially U. parvum) became friendlier than their ancestors to their human host(by losing most of its virulence genes); however, GBS tried to increase its invasiveabilities (by getting more virulence genes) to fight against the human host attack

Using a practical molecular capsular serotype prediction strategy to investigate Streptococcus pneumoniae serotype distribution and antimicrobial resistance in Chinese local hospitalized children

Comprehensive cpsB sequetyping database based on GenBank sequences (until Jan 1, 2015). (DOCX 47 kb

A Personalized Rolling Optimal Charging Schedule for Plug-In Hybrid Electric Vehicle Based on Statistical Energy Demand Analysis and Heuristic Algorithm

To alleviate the emission of greenhouse gas and the dependence on fossil fuel, Plug-in Hybrid Electrical Vehicles (PHEVs) have gained an increasing popularity in current decades. Due to the fluctuating electricity prices in the power market, a charging schedule is very influential to driving cost. Although the next-day electricity prices can be obtained in a day-ahead power market, a driving plan is not easily made in advance. Although PHEV owners can input a next-day plan into a charging system, e.g., aggregators, day-ahead, it is a very trivial task to do everyday. Moreover, the driving plan may not be very accurate. To address this problem, in this paper, we analyze energy demands according to a PHEV owner’s historical driving records and build a personalized statistic driving model. Based on the model and the electricity spot prices, a rolling optimization strategy is proposed to help make a charging decision in the current time slot. On one hand, by employing a heuristic algorithm, the schedule is made according to the situations in the following time slots. On the other hand, however, after the current time slot, the schedule will be remade according to the next tens of time slots. Hence, the schedule is made by a dynamic rolling optimization, but it only decides the charging decision in the current time slot. In this way, the fluctuation of electricity prices and driving routine are both involved in the scheduling. Moreover, it is not necessary for PHEV owners to input a day-ahead driving plan. By the optimization simulation, the results demonstrate that the proposed method is feasible to help owners save charging costs and also meet requirements for driving

Diversity of group B streptococcus serotypes causing urinary tract infection in adults

Serotypes of group B streptococcus (GBS) that cause urinary tract infection (UTI) are poorly characterized. We conducted a prospective study of GBS UTI in adults to define the clinical and microbiological characteristics of these infections, including which serotypes cause disease. Patients who had GBS cultured from urine over a 1-year period were grouped according to symptoms, bacteriuria, and urinalysis. Demographic data were obtained by reviewing medical records. Isolates were serotyped by latex agglutination and multiplex PCRreverse line blotting (mPCR/RLB). Antibiotic susceptibilities were determined by disc diffusion. GBS was cultured from 387/34,367 consecutive urine samples (1.1%): 62 patients had bacteriuria of >10 7 CFU/liter and at least one UTI symptom; of these patients, 31 had urinary leukocyte esterase and pyuria (others not tested), 50 (81%) had symptoms consistent with cystitis, and 12 (19%) had symptoms of pyelonephritis. Compared with controls (who had GBS isolated without symptoms), a prior history of UTI was an independent risk factor for disease. Increased age was also significantly associated with acute infection. Serotyping results were consistent between latex agglutination and mPCR/RLB for 331/387 (85.5%) isolates; 22 (5.7%) and 7 (1.8%) isolates were nontypeable with antisera and by mPCR/RLB, respectively; and 45/56 (80.4%) isolates with discrepant results were typed by mPCR/RLB as belonging to serotype V. Serotypes V, Ia, and III caused the most UTIs; serotypes II, Ib, and IV were less common. Nontypeable GBS was not associated with UTI. Erythromycin (39.5%) and clindamycin (26.4%) resistance was common. We conclude that a more diverse spectrum of GBS serotypes causes UTI than previously recognized, with the exception of nontypeable GBS

Contributions of aromatic pairs of human Gamma-D-Crystallin to its folding, stability, aggregation, and interaction with human Alpha B-Crystallin

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2012.Cataloged from PDF version of thesis. Vita.Includes bibliographical references (p. 139-158).Two distinct groups of proteins, a-crystallins and [Beta][gamma]-crystallins, constitute 90% of the vertebrate eye lens soluble proteins. Long-term solubility and stability against unfolding and aggregation are essential properties of crystallins and crucial to the function of the lens. Aggregation of crystallins in the lens causes light scattering and directly contributes to development of cataract, the leading cause of blindness in the world. The amino acid determinants of these biochemical/biophysical properties of crystallins are not entirely understood. Aromatic residues in proteins have been shown to be important determinants of their folding pathways, native-state stability, aggregation propensity and other intermolecular interactions. In this thesis study, I have investigated the contributions of the paired aromatic residues of human yD-crystallin (H[gamma]D-Crys) to its folding, stability, aggregation, and interaction with the chaperone human aB-crystallin (H[alpha][Beta]-Crys). H[gamma]D-Crys is a highly stable protein that remains folded in the nucleus of the eye lens for the majority of an individual's lifetime. Like other [Beta][gamma]-crystallins, H[gamma]D-Crys exhibits two homologous crystallin domains, each containing two Greek key motifs and eight [Beta]-strands. Six conserved aromatic pairs (four Tyr/Tyr, one Tyr/Phe and one Phe/Phe) are present in H[gamma]D-Crys. Four among them are located at conserved [Beta]-hairpins of the Greek key motifs, thus termed "Greek key pairs". The Greek key pairs have the consensus sequence Y/FXXXXY/FXG and are one of the defining features of the [Beta][[gamma]-crystallin family. Ultraviolet (UV) damage to these aromatic residues in [Beta][gamma]-crystallins may contribute to unfolding and aggregation of the proteins, leading to development of cataract. [Alpha]-Crystallins belong to the small heat shock protein (sHsp) family and have both structural and chaperone functions in the lens. Human a-crystallins form polydisperse oligomers of 15-60 subunits, with aA:aB ratio about 3:1 in vivo. The core a-crystallin domain (aCD) of acrystallins has an immunoglobulin (Ig)-like p-sandwich fold, but the quaternary structure of acrystallin remains to be fully solved. Like other sHsps, a-crystallins exert their chaperone function by sequestering partially-unfolded and aggregation-prone substrates in an ATPindependent manner, thus preventing their aggregation. The chaperone-substrate interactions of a-crystallins and other sHsps remain poorly understood. To investigate the roles of the paired aromatic residues in H[gamma]D-Crys, mutant proteins with these aromatic residues substituted with alanines were constructed and expressed in E. coli. All mutant proteins maintained native-like secondary structures by circular dichroism (CD). Except F 115A and F 117A, all mutant proteins had lower thermal stability than the wildtype (WT) protein. Equilibrium unfolding/refolding experiments in guanidine hydrochloride (GuHCl) showed that all mutant proteins had lower thermodynamic stability than the WT protein. Nterminal domain (N-td) substitutions shifted the N-td transitions to lower GuHCl concentrations, but the C-terminal domain (C-td) transitions remained unaffected. C-td substitutions led to a more synchronized unfolding/refolding process of the N-td and C-td, and the overall transitions shifted to lower GuHCl concentrations. These results were consistent with a sequential unfolding/refolding model of H[gamma]D-Crys, in which the N-td unfolds first and refolds last. The Greek key pairs had larger contributions to both thermal stability and thermodynamic stability than the non-Greek-key pairs. Aromatic-aromatic interaction energy was estimated by double mutant cycles as 1.5-2.0 kcal/mol. To distinguish the effects in unfolding and refolding, kinetic experiments were also performed. In kinetic unfolding experiments, N-td substitutions accelerated the early phase of unfolding, while C-td substitutions accelerated the late phase. For refolding, only substitutions of the second Greek key pair of each crystallin domain slowed refolding: N-td substitutions Y45A and Y5OA affected the late phase while Y133A and Y138A affected the early phase of the overall refolding reaction. The second Greek key may serve as a nucleation site during the folding of the double-Greek-key crystallin domain. The aggregation pathway that competes with productive refolding in vitro, as well as the suppression of aggregation by chaperone H[alpha]B-Crys were also investigated for mutant H[gamma]D-Crys variants with replacements of the Greek key paired residues. The WT and the mutant H[gamma]D-Crys behaved very similarly, in term of aggregation kinetics and final aggregation level, indicating that these aromatic pairs played minimal role in the aggregation process. The efficiencies of aggregation suppression by H[alpha]B-Crys, as well as the H[alpha]B-bound conformations characterized by the fluorescence of H[gamma]B-H[gamma]D complexes were also very similar for the WT and mutant HyDCrys, arguing against a critical role of these aromatic pairs in the chaperone recognition process. Together with the earlier results, it can also be concluded that stability and refolding kinetics of H[gamma]D-Crys were not critical determinants for its refolding-induced aggregation as well as HaBCrys recognition. Both of these processes may rely on features of the folding intermediate in a very early stage of Greek key refolding. The tryptophan fluorescence of the H[alpha]B-H[gamma]D complexes with WT or mutant H[gamma]D-Crys resembled a partially unfolded state of HyD-Crys, consistent with the tryptophans being part of the contact sites with the chaperone H[alpha]B-Crys.by Fanrong Kong.Ph.D

Investigation of Thermal Cycle and Hardness Distribution in the Laser Cladding of AISI H13 tool steel produced by a High Power Direct Diode Laser

Laser cladding (LC) of tool steel has significant application in rapid tooling, and surface coating for worn-out components in different industries. During the LC process, several phase transformations influence the microstructural and mechanical properties of the deposited layer. In order to successfully implement the LC process, it is essential to understand the relationship between the thermal cycle (heating and cooling), phase transformations, and the output quantities of the deposited layer. In this study a direct diode laser with a power of up to 8 kW was used to deposit AISI H13 tool steel on mild steel grade A36 substrate to enhance its surface properties. Primarily, an experimentally verified three-dimensional (3-D) heat transfer analysis was developed based on the finite element method to compute temperature history during the cladding and cooling process. Next, the computed thermal cycles were coupled with a semi-empirical thermo-kinetic model to estimate the hardness of deposited layers based on different cooling cycles in a time-temperature-transformation (TTT) diagram. Further, the microstructural details obtained from the cross-sections of the clad were correlated with the estimated thermal cycles and hardness. A good correlation between the modeled and experimental results revealed that the developed model can be used to estimate the microstructural characteristics and mechanical properties of the H13 layer produced by the LC process

Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction followed by probe hybridization on a nylon membrane, which is re-usable. Probes are 5' amine modified to allow fixation to the membrane. Primers are 5' biotin modified which allows detection of hybridized PCR products using streptavidin-peroxidase and a chemiluminescent substrate via photosensitive film. With low setup and consumable costs, this technique is inexpensive (approximately US$2 per sample), high throughput (multiple membranes can be processed simultaneously) and has a short turnaround time (approximately 10 hours)