Conjugative plasmid-encoded toxin-antitoxin system PrpT/PrpA directly controls plasmid copy number.

Toxin-antitoxin (TA) loci were initially identified on conjugative plasmids, and one function of plasmid-encoded TA systems is to stabilize plasmids or increase plasmid competition via postsegregational killing. Here, we discovered that the type II TA system, Pseudoalteromonas rubra plasmid toxin-antitoxin PrpT/PrpA, on a low-copy-number conjugative plasmid, directly controls plasmid replication. Toxin PrpT resembles ParE of plasmid RK2 while antitoxin PrpA (PF03693) shares no similarity with previously characterized antitoxins. Surprisingly, deleting this prpA-prpT operon from the plasmid does not result in plasmid segregational loss, but greatly increases plasmid copy number. Mechanistically, the antitoxin PrpA functions as a negative regulator of plasmid replication, by binding to the iterons in the plasmid origin that inhibits the binding of the replication initiator to the iterons. We also demonstrated that PrpA is produced at a higher level than PrpT to prevent the plasmid from overreplicating, while partial or complete degradation of labile PrpA derepresses plasmid replication. Importantly, the PrpT/PrpA TA system is conserved and is widespread on many conjugative plasmids. Altogether, we discovered a function of a plasmid-encoded TA system that provides new insights into the physiological significance of TA systems.

Parallel analysis of RNA ends reveals global microRNA-mediated target RNA cleavage in maize.

MicroRNAs (miRNAs) are endogenous 20-24-nucleotide non-coding RNAs that play important regulatory roles in many biological processes in eukaryotes. miRNAs modulate the expression of target genes at the post-transcriptional level by transcript cleavage or translational inhibition. The identification of miRNA target genes has been extensively investigated in Arabidopsis and rice, but an in-depth global analysis of miRNA-mediated target regulation is still lacking in maize. Here, we report a transcriptome-wide identification of miRNA targets by analyzing parallel analysis of RNA ends (PARE) datasets derived from nine different tissues at five developmental stages of the maize (Zea mays L.) B73 cultivar. In total, 246 targets corresponding to 60 miRNAs from 25 families were identified, including transcription factors and other genes. In addition, PARE analysis revealed that miRNAs guide specific target transcript cleavage in a tissue-preferential manner. Primary transcripts of MIR159c and MIR169e were found to be cleaved by mature miR159 and miR169, respectively, indicating a negative-feedback regulatory mechanism in miRNA biogenesis. Moreover, several miRNA-target gene pairs involved in seed germination were identified and experimentally validated. Our PARE analyses generated a wide and detailed miRNA-target interaction atlas, which provides a valuable resource for investigating the roles of miRNAs and their targets in maize.

Small RNAs target native and cross-kingdom transcripts on both sides of the wheat stripe rust interaction.

The wheat stripe rust fungus (Puccinia striiformis f.sp. tritici) threatens global wheat production. Small RNAs (sRNAs) modulate plant defense induction, and RNA exchange between host and microbe causes cross-kingdom gene silencing, but few examples are known in rust fungi. This study combined sRNA, parallel analysis of RNA ends, and gene expression data to discover sRNA-target pairs on each side of the interaction. Specific wheat 24 nt sRNAs were suppressed, while particular 35 nt fragments were strongly induced upon infection. Wheat sRNAs cleaved fungal transcripts coding for a ribosomal protein and a glycosyl hydrolase effector. Fungal microRNA-like and phased 21 nt sRNAs originated from long inverted repeats near protein coding genes. Fungal sRNAs targeted native transcripts: transposons and kinases; and cross-kingdom transcripts: a wheat nucleotide-binding domain leucine-rich repeat receptor (NLR) and multiple defense-related transcription factor families. This work sheds light on host-microbe coevolution and delivers prospects for developing pathogen control biotechnology.

Management of injuries on the 16th-century battlefield: Ambroise Paré's contributions to neurosurgery and functional recovery.

During the 1536 siege of Turin in northern Italy, a young French barber-surgeon abandoned the conventional treatment of battle-inflicted wounds, launching a revolution in military medicine and surgical techniques. Ambroise Paré (1510-1590) was born into a working-class Huguenot family in Laval, France, during an era when surgery was not considered a respectable profession. He rose from humble origins as a barber-surgeon, a low-ranked occupation in the French medical hierarchy, to become a royal surgeon (chirurgien ordinaire du Roi) serving 4 consecutive French monarchs. His innovative ideas and surgical practice were a response to the environment created by new military technology on 16th-century European battlefields. Gunpowder weapons caused unfamiliar, complicated injuries that challenged Paré to develop new techniques and surgical instruments. Although Paré's contributions to the treatment of wounds and functional prosthetics are documented, a deeper appreciation of his role in military neurosurgery is needed. This paper examines archives, primary texts, and written accounts by Paré that reveal specific patient cases highlighting his innovative contributions to neurotrauma and neurosurgery during demanding and harrowing circumstances, on and off the battlefield, in 16th-century France. Notably, trepanation indications increased because of battlefield head injuries, and Paré frequently described this technique and improved the design of the trepan tool. His contribution to neurologically related topics is extensive; there are more chapters devoted to the nervous system than to any other organ system in his compendium, Oeuvres. Regarding anatomical knowledge as fundamentally important and admiring the contemporary contributions of Andreas Vesalius, Paré reproduced many images from Vesalius' works at his own great expense. The manner in which Paré's participation in military expeditions enabled collaboration with multidisciplinary artisans on devices, including surgical tools and prosthetics, to restore neurologically associated functionality is also discussed. Deeply religious, in a life filled with adventure, and serving in often horrendous conditions during a time when Galenic dogma still dominated medical practice, Paré developed a reputation for logic, empiricism, technology, and careful treatment. "I have [had] the opportunity to praise God, for what he called me to do in medical operation, which is commonly called surgery, which could not be bought with gold or silver, but by only virtue and great experimentation."

Degradome Analysis of Tomato and Nicotiana benthamiana Plants Infected with Potato Spindle Tuber Viroid.

Viroids are infectious non-coding RNAs that infect plants. During infection, viroid RNAs are targeted by Dicer-like proteins, generating viroid-derived small RNAs (vd-sRNAs) that can guide the sequence specific cleavage of cognate host mRNAs via an RNA silencing mechanism. To assess the involvement of these pathways in pathogenesis associated with nuclear-replicating viroids, high-throughput sequencing of sRNAs and degradome analysis were carried out on tomato and Nicotiana benthamiana plants infected by potato spindle tuber viroid (PSTVd). Both hosts develop similar stunting and leaf curling symptoms when infected by PSTVd, thus allowing comparative analyses. About one hundred tomato mRNAs potentially targeted for degradation by vd-sRNAs were initially identified. However, data from biological replicates and comparisons between mock and infected samples reduced the number of bona fide targets-i.e., those identified with high confidence in two infected biological replicates but not in the mock controls-to only eight mRNAs that encode proteins involved in development, transcription or defense. Somewhat surprisingly, results of RT-qPCR assays revealed that the accumulation of only four of these mRNAs was inhibited in the PSTVd-infected tomato. When these analyses were extended to mock inoculated and PSTVd-infected N. benthamiana plants, a completely different set of potential mRNA targets was identified. The failure to identify homologous mRNA(s) targeted by PSTVd-sRNA suggests that different pathways could be involved in the elicitation of similar symptoms in these two species. Moreover, no significant modifications in the accumulation of miRNAs and in the cleavage of their targeted mRNAs were detected in the infected tomato plants with respect to the mock controls. Taken together, these data suggest that stunting and leaf curling symptoms induced by PSTVd are elicited by a complex plant response involving multiple mechanisms, with RNA silencing being only one of the possible components.

MicroRNA-guided regulation of heat stress response in wheat.

BACKGROUND: With rising global temperature, understanding plants' adaptation to heat stress has implications in plant breeding. MicroRNAs (miRNAs) are small, non-coding, regulatory RNAs guiding gene expression at the post-transcriptional level. In this study, small RNAs and the degradome (parallel analysis of RNA ends) of leaf tissues collected from control and heat-stressed wheat plants immediately at the end of the stress period and 1 and 4 days later were analysed. RESULTS: Sequencing of 24 small RNA libraries produced 55.2 M reads while 404 M reads were obtained from the corresponding 24 PARE libraries. From these, 202 miRNAs were ascertained, of which mature miRNA evidence was obtained for 104 and 36 were found to be differentially expressed after heat stress. The PARE analysis identified 589 transcripts targeted by 84 of the ascertained miRNAs. PARE sequencing validated the targets of the conserved members of miRNA156, miR166 and miR393 families as squamosa promoter-binding-like, homeobox leucine-zipper and transport inhibitor responsive proteins, respectively. Heat stress responsive miRNA targeted superoxide dismutases and an array of homeobox leucine-zipper proteins, F-box proteins and protein kinases. Query of miRNA targets to interactome databases revealed a predominant association of stress responses such as signalling, antioxidant activity and ubiquitination to superoxide dismutases, F-box proteins, pentatricopeptide repeat-containing proteins and mitochondrial transcription termination factor-like proteins. CONCLUSION: The interlaced data set generated in this study identified and validated heat stress regulated miRNAs and their target genes associated with thermotolerance. Such accurate identification and validation of miRNAs and their target genes are essential to develop novel regulatory gene-based breeding strategies.

Prevalence of fluoroquinolone resistance and mutations in the gyrA, parC and parE genes of Riemerella anatipestifer isolated from ducks in China.

BACKGROUND: Riemerella anatipestifer is one of the most serious infectious disease-causing pathogens in the duck industry. Drug administration is an important method for prevention and treatment of infection in duck production, leading to widespread drug resistance in R. anatipestifer. METHODS: For a total of 162 isolates of R. anatipestifer, the MICs were determined for a quinolone antimicrobial agent, namely, nalidixic acid, and three fluoroquinolones, namely, ciprofloxacin, enrofloxacin and ofloxacin. The gyrA, parC, and parE gene fragments were amplified by PCR to identify the mutation sites in these strains. Site-directed mutants with mutations that were detected at a high frequency in vivo were constructed (hereafter referred to as site-directed in vivo mutants), and the MICs of these four drugs for these strains were determined. RESULTS: In total, 100, 97.8, 99.3 and 97.8% of the 137 R. anatipestifer strains isolated between 2013 and 2018 showed resistance to nalidixic acid, ciprofloxacin, enrofloxacin, and ofloxacin, respectively. The high-frequency mutation sites were detected in a total of 162 R. anatipestifer strains, such as Ser83Ile and Ser83Arg, which are two types of substitution mutations of amino acid 83 in GyrA; Val799Ala and Ile811Val in ParC; and Val357Ile, His358Tyr, and Arg541Lys in ParE. MIC analysis results for the site-directed in vivo mutants showed that the strains with only the Ser83Ile mutation in GyrA exhibited an 8-16-fold increase in MIC values, and all mutants showed resistance to ampicillin and ceftiofur. CONCLUSIONS: The resistance of R. anatipestifer to quinolone agents is a serious problem. Amino acid 83 in GyrA is the major target mutation site for the fluoroquinolone resistance mechanism of R. anatipestifer.

Congenital orthopaedic limp deformities in Corpus Hippocraticum.

During the fifth century BC in ancient Greece during the eve of orthopaedics, the Hippocratic School of Medicine diagnosed a series of congenital limb deformities. Congenital dislocation of the arm, elbow, wrist, hip, knee, tarsotibial joint, apex leg, as well as talipes valgus (clubfoot), congenital clavicle fractures, and thumb malfunction were all discussed by Hippocrates and his followers. Ancient Greek medico-philosophers, fond of a "perfect" human body, proposed an immediate non-interventional approach, while archaic orthotics and specialized footwear were suggested. The Hippocratic methodology was once more re-emerged in the sixteenth century by Ambroise Paré and in the nineteenth century by Wilhelm Roser, becoming since then the main principle for the confrontation of congenital deformities. Various surgeons until nowadays are still being influenced by the Hippocratic doctrine.

Towards Exploring Toxin-Antitoxin Systems in Geobacillus: A Screen for Type II Toxin-Antitoxin System Families in a Thermophilic Genus.

The toxin-antitoxin (TA) systems have been attracting attention due to their role in regulating stress responses in prokaryotes and their biotechnological potential. Much recognition has been given to type II TA system of mesophiles, while thermophiles have received merely limited attention. Here, we are presenting the putative type II TA families encoded on the genomes of four Geobacillus strains. We employed the TA finder tool to mine for TA-coding genes and manually curated the results using protein domain analysis tools. We also used the NCBI BLAST, Operon Mapper, ProOpDB, and sequence alignment tools to reveal the geobacilli TA features. We identified 28 putative TA pairs, distributed over eight TA families. Among the identified TAs, 15 represent putative novel toxins and antitoxins, belonging to the MazEF, MNT-HEPN, ParDE, RelBE, and XRE-COG2856 TA families. We also identified a potentially new TA composite, AbrB-ParE. Furthermore, we are suggesting the Geobacillus acetyltransferase TA (GacTA) family, which potentially represents one of the unique TA families with a reverse gene order. Moreover, we are proposing a hypothesis on the xre-cog2856 gene expression regulation, which seems to involve the c-di-AMP. This study aims for highlighting the significance of studying TAs in Geobacillus and facilitating future experimental research.

Analysis of Brachypodium miRNA targets: evidence for diverse control during stress and conservation in bioenergy crops.

BACKGROUND: Since the proposal of Brachypodium distachyon as a model for the grasses, over 500 Bdi-miRNAs have been annotated in miRBase making Brachypodium second in number only to rice. Other monocots, such as switchgrass, are completely absent from the miRBase database. While a significant number of miRNAs have been identified which are highly conserved across plants, little research has been done with respect to the conservation of miRNA targets. Plant responses to abiotic stresses are regulated by diverse pathways many of which involve miRNAs; however, it can be difficult to identify miRNA guided gene regulation when the miRNA is not the primary regulator of the target mRNA. RESULTS: To investigate miRNA target conservation and stress response involvement, a set of PARE (Parallel Analysis of RNA Ends) libraries totaling over two billion reads was constructed and sequenced from Brachypodium, switchgrass, and sorghum representing the first report of RNA degradome data from the latter two species. Analysis of this data provided not only PARE evidence for miRNA guided cleavage of over 7000 predicted target mRNAs in Brachypodium, but also evidence for miRNA guided cleavage of over 1000 homologous transcripts in sorghum and switchgrass. A pipeline was constructed to compare RNA-seq and PARE data made from Brachypodium plants exposed to various abiotic stress conditions. This resulted in the identification of 44 miRNA targets which exhibit stress regulated cleavage. Time course experiments were performed to reveal the relationship between miR393ab, miR169a, miR394ab, and their respective targets throughout the first 36 h of the cold stress response in Brachypodium. CONCLUSIONS: Knowledge gained from this study provides considerable insight into the RNA degradomes and the breadth of miRNA target conservation among these three species. Additionally, associations of a number of miRNAs and target mRNAs with the stress responses have been revealed which could aid in the development of stress tolerant transgenic crops.

Ambroise Paré (1510-1590): on the diagnosis and treatment of shoulder dislocations.

AIM OF STUDY: This article aims to summarize the treatment methods for shoulder dislocations supported by Ambroise Paré, and his clinical and academic contribution to this subject. INTRODUCTION: Ambroise Paré (1510-1590) was one of the most prominent surgeons of his time, and one of the most significant innovators of the Renaissance in the fields of military operation, bandages, wound healing, bloodletting, close blood vessels techniques and cesarean section. For many, he is considered "the Father of Modern Surgery". METHODS: In our search we went through the literature in Pubmed and Google Scholar as well as the works of Hippocrates and Galen and several other ancient and medieval authors. RESULTS: His work on shoulder injuries and dislocations is outstanding. In this work, he uses the classifications of Hippocrates (460-377 BC) and Galen (130-201 BC). He also uses the reduction techniques, ointments, bandages, cooling creams and treatment options described by the ancient medical authors. DISCUSSION: Paré is heavily based on the work of previous authors on shoulder dislocations. He uses the work of Hippocrates and Galen as well as the additions made in the Cannon of Medicne of Avicenna. CONCLUSION: Ambroise Paré in his excellent work on shoulder dislocations uses the classifications of Hippocrates and Galen and the reduction techniques, cooling ointments, bandages and post-reduction management options as previously described by Hippocrates himself.

Pathfinders in oncology from the end of the Middle Ages to the beginning of the 19th century.

During the 3 centuries reviewed herein, all that was known regarding cancer was limited to Italy, France, Germany, and England. Practically nothing was written about cancer in the rest of the known world, including the newly discovered geographic areas in the Americas, Africa, and Australia. The 7 pioneer physicians considered in this review lived through some of the most turbulent geopolitical, social, and religious upheavals that Europe had experienced during the Renaissance. Despite their diverse social, political, and religious backgrounds and disparate personalities, they were united in their commitment to the advancement of medicine and oncology and to spreading knowledge through printed texts. Collectively, they established the groundwork for modern anatomy, physiology, chemistry, pathology, surgical oncology, and chemotherapy. It perhaps is noteworthy that 5 of the 7 physicians who contributed the most to the advancement of surgery and surgical oncology served in various wars as army surgeons. Considering everything together, the refusal of physicians of this epoch to use the microscope is a historical enigma. Cancer 2017;123:1888-1897. © 2017 American Cancer Society.

A RelE/ParE superfamily toxin in Vibrio parahaemolyticus has DNA nicking endonuclease activity.

Type II toxins in toxin-antitoxin (TA) systems fold into a similar fold and belong to the RelE/ParE superfamily. However, they display two distinct biochemical activities: RelE toxins are mRNA interferases, while ParE toxins are DNA gyrase (Gyr) inhibitors. Previously, we found a TA system, vp1842/vp1843, on the Vibrio parahaemolyticus genome whose toxin Vp1843 belongs to the RelE/ParE toxin superfamily. Vp1843, unlike RelE toxins, has neither protein synthesis inhibitory activity nor ribonuclease activity. In this study, we examined the inhibitory potency of Vp1843 with Escherichia coli Gyr. The result showed that Vp1843, unlike other ParE toxins, had little Gyr inhibitory activity, but rather converted supercoiled DNA to open-circular DNA. Analysis showed further that Vp1843 cleaves a single strand in DNA, and that the antitoxin Vp1842 neutralized the nicking endonuclease activity of Vp1843. Mutations of Lys37 and Pro45 in Vp1843 abolished its nicking activity, suggesting that they play a crucial role in nicking endonuclease activity. To our knowledge, Vp1843 is the first toxin with DNA nicking endonuclease activity among the RelE/ParE toxin superfamily.

Small RNA and PARE sequencing in flower bud reveal the involvement of sRNAs in endodormancy release of Japanese pear (Pyrus pyrifolia 'Kosui').

BACKGROUND: In woody perennial plants, including deciduous fruit trees, such as pear, endodormancy is a strategy for surviving the cold winter. A better understanding of the mechanism underlying the endodormancy phase transition is necessary for developing countermeasures against the effects of global warming. In this study, we analyzed the sRNAome of Japanese pear flower buds in endodormant and ecodormant stages over two seasons by implementing of RNA-seq and degradome-sequencing. RESULTS: We identified 137 conserved or less conserved miRNAs and 50 pear-specific miRNAs. However, none of the conserved microRNAs or pear-specific miRNAs was differentially expressed between endodormancy and ecodormancy stages. On the contrast, 1540 of 218,050 loci that produced sRNAs were differentially expressed between endodormancy and ecodormancy, suggesting their potential roles on the phase transition from endodormancy to ecodomancy. We also characterized a multifunctional miRNA precursor MIR168, which produces two functional miR168 transcripts, namely miR168.1 and miR168.2; cleavage events were predominantly mediated by the non-conserved variant miR168.2 rather than the conserved variant miR168.1. Finally, we showed that a TAS3 trans-acting siRNA triggered phased siRNA within the ORF of one of its target genes, AUXIN RESPONSE FACTOR 4, via the analysis of phased siRNA loci, indicating that siRNAs are able to trigger phased siRNAs in pear. CONCLUSION: We analyzed the sRNAome of pear flower bud during dormant phase transition. Our work described the sRNA profiles of pear winter buds during dormant phase transition, showing that dormancy release is a highly coordinated physiological process involving the regulation of sRNAs.

Two cases of 16th century head injuries managed in royal European families.

In Europe, during the 16th century, there were a number of prominent general surgeons adventurous enough to consider operating on the brain for head injuries. From the time of Hippocrates, operating on the skull and brain was considered both treacherous and too dangerous to be undertaken except on rare occasions. Operating on a member of a royal court was considered even more exceptional because if the outcome was poor, the surgeon could lose a hand or limb, or, even worse, be beheaded. The authors present two interesting cases of royal family members who underwent surgery for head injuries that were quite severe. The surgeons involved, Ambroise Paré, Andreas Vesalius, and Berengario da Carpi, were among the most prominent surgeons in Europe. Despite very challenging political situations, all were willing to undertake a complex surgical intervention on the member of a prominent royal family. The individuals involved, both royal and medical, plus the neurosurgical injuries are discussed.

Differential expression of miRNAs and their target genes in senescing leaves and siliques: insights from deep sequencing of small RNAs and cleaved target RNAs.

MicroRNAs (miRNAs) are a class of small RNAs, which typically function by guiding cleavage of target mRNAs. They are known to play roles in a variety of plant processes including development, responses to environmental stresses and senescence. To identify senescence regulation of miRNAs in Arabidopsis thaliana, eight small RNA libraries were constructed and sequenced at four different stages of development and senescence from both leaves and siliques, resulting in more than 200 million genome-matched sequences. Parallel analysis of RNA ends libraries, which enable the large-scale examination of miRNA-guided cleavage products, were constructed and sequenced, resulting in over 750 million genome-matched sequences. These large datasets led to the identification a new senescence-inducible small RNA locus, as well as new regulation of known miRNAs and their target genes during senescence, many of which have established roles in nutrient responsiveness and cell structural integrity. In keeping with remobilization of nutrients thought to occur during senescence, many miRNAs and targets had opposite expression pattern changes between leaf and silique tissues during the progression of senescence. Taken together, these findings highlight the integral role that miRNAs may play in the remobilization of resources and alteration of cellular structure that is known to occur in senescence.

Rapid construction of parallel analysis of RNA end (PARE) libraries for Illumina sequencing.

MicroRNAs (miRNAs) are ∼21nt small RNAs that pair to their target mRNAs and in many cases trigger cleavage, particularly in plants. Although many computational tools can predict miRNA:mRNA interactions, it remains critical to validate cleavage events, due to miRNA function in translational repression or due to high rates of false positives (over 90%) for unvalidated target predictions. A few years ago, three laboratories described similar methods to validate cleavage of miRNA targets by the cloning en masse of 5' ends of cleaved or uncapped mRNAs. To take advantage of the recent progress in high-throughput sequencing technology, we have devised an updated protocol to (1) enable much faster library preparation, and (2) reduce the cost by pooling indexed samples together for sequencing. Here we provide a step-by-step protocol for PARE library construction, starting from total RNA. This protocol has been successfully used in our laboratory to validate miRNA targets in a variety of plant species. We also provide advice for troubleshooting on some common issues.

Dual targeting DNA gyrase B (GyrB) and topoisomerse IV (ParE) inhibitors: A review.

GyrB and ParE are type IIA topoisomerases and found in most bacteria. Its function is vital for DNA replication, repair and decatenation. The highly conserved ATP-binding subunits of DNA GyrB and ParE are structurally related and have been recognized as prime candidates for the development of dual-targeting antibacterial agents with broad-spectrum potential. However, no natural product or small molecule inhibitors targeting ATPase catalytic domain of both GyrB and ParE enzymes have succeeded in the clinic. Moreover, no inhibitors of these enzymes with broad-spectrum antibacterial activity against Gram-negative pathogens have been reported. Availability of high resolution crystal structures of GyrB and ParE made it possible for the design of many different classes of inhibitors with dual mechanism of action. Among them benzimidazoles, benzothiazoles, thiazolopyridines, imidiazopyridazoles, pyridines, indazoles, pyrazoles, imidazopyridines, triazolopyridines, pyrrolopyrimidines, pyrimidoindoles as well as related structures are disclosed in literatures. Unfortunately most of these inhibitors are found to be active against Gram-positive pathogens. In the present review we discuss about studies on novel dual targeting ATPase inhibitors.

Dominance of clonal complex 10 among the levofloxacin-resistant Streptococcus agalactiae isolated from bacteremic patients in a Korean hospital.

Streptococcus agalactiae has emerged as an important cause of invasive infection in adults. Forty-nine S. agalactiae isolates (41 from adults and 8 from neonates) were collected during a 4-year period (2010-2013) and analyzed by multilocus sequence typing (MLST). Antibiotic susceptibility to erythromycin, clindamycin and levofloxacin was determined and the determinants of resistance (ermA, ermB, ermC, mefA, lnuB) were detected by PCR and mutation in gyrA, gyrB, parC and parE gene was investigated by sequence analysis. They were resolved into 14 sequence types (STs) and belonged to five clonal complexes (CCs). The distribution of CC was significantly different according to the age group; CC1 (18/41) and CC10 (13/41) was the most common among the adult isolates but CC19 (5/8) was predominant among the neonatal isolates. The resistance rate to erythromycin, clindamycin was 18.4% and 24.5%, respectively. Among the 13 strains resistant to erythromycin and/or clindamycin, two isolates harbored ermA and 10 isolates harbored ermB. The levofloxacin resistance rate was very high (32.7%) and was significantly higher in CC10 (71.4%). All the levofloxacin-resistant isolates had identical gyrA substitution (Ser81Leu) but parC substitution was different according to the CCs. The additional mutation in parE (His221Tyr) was found only in CC19. Continuous monitoring of the fluoroquinolone resistance and genotypic distribution among S. agalactiae is needed.

Renaissance-Europe-16th century.

Realistic images became available for the first time. The first major figure was Berengario da Carpi (1460-1530). He made contributions to knowledge. He stated the dura was attached all over the interior of the cranium not just at the sutures. He also noted that deterioration following traumatic hematomas was speedier the deeper within the brain the bleed had occurred and he noticed that post-traumatic neurological deficits were contralateral. Moreover, he introduced new instruments of a practical design. Specifically, he launched trepanation using a brace and bit handle. This instrument required two hands and rotated the trepan in the same direction all the time. In addition, he illustrated a crown trepan in which the bits could be interchanged. He also developed an improved elevator. He also provided the first drawing of a lenticular. The next illustrations came from Vidus Vidius (1509-1569). The illustrations in his text were elegant and realistic but some of them were impractical or unusable. Ambroise Paré (1510-1590) was a major surgeon. He designed an improved brace and bit trepan with a collar to control penetration. He also introduced instruments for expanding a cranial opening by biting up the bone and for depressing the dura to enable material to escape more easily.