Simultaneous Electrochemical Detection of Multiple Bacterial Species Using Metal-Organic Nanohybrids.

Organic metallic nanohybrids (NHs), in which many small metal nanoparticles are encapsulated within a conductive polymer matrix, are useful as sensitive electrochemical labels because the constituents produce characteristic oxidation current responses. Gold NHs, consisting of gold nanoparticles and poly(m-toluidine), and copper NHs, consisting of copper nanoparticles and polyaniline, did not interfere with each other in terms of the electrochemical signals obtained on the same electrode. Antibodies were introduced into these NHs to function as electrochemical labels for targeting specific bacteria. Electrochemical measurements using screen-printed electrodes dry-fixed with NH-labeled bacterial cells enabled the estimation of bacterial species and number within minutes, based on the distinct current response of the labels. Our proposed method achieved simultaneous detection of enterohemorrhagic Escherichia coli and Staphylococcus aureus in a real sample. These NHs will be powerful tools as electrochemical labels and are expected to be useful for rapid testing in food and drug-related manufacturing sites.

Effectiveness of Clinical Management of COVID-19 Based on Structured Clinical Knowledge and Process Paths.

This study examined the effectiveness of a systematic approach to the clinical management of COVID-19, focusing on nursing turnover. METHODS: Between 2017 and 2019, a clinical process support system based on structured clinical knowledge (Team Compass with the Patient Condition Adaptive Path System; TC-PCAPS) was developed, and implemented in hospitals. In 2020, the COVID-19 clinical management system (COVID-19-CMS) was developed. In this study, the effectiveness of implementing both systems was analyzed. The analysis covered hospitals N, T, and B, where TC-PCAPS implementation started in 2019, 2020, and 2022, respectively. Data for the period from 2018 to 2022 were collected and compared. RESULTS: Hospitals N and T implemented TC-PCAPS in the first year and the COVID-19-CMS in the following year. The nurse turnover rates of these hospitals were lower than those of the prefectures in which they were located. There was a trend towards a gradual reduction in nurse turnover. In contrast, hospital B, which had only just started to introduce these systems, saw a gradual increase in nurse turnover. CONCLUSION: The data collected from these three hospitals suggested that this systematic approach has the potential to reduce nurse turnover, in addition to the previously reported ability of TC-PCAPS to reduce nurse overtime. In Japan, there is a need to respond to future pandemics and reform the work styles of physicians and nurses. The abovementioned systematic approach has great potential for contributing to both of these aims.

Patient Data Sharing and Reduction of Overtime Work of Nurses by Innovation of Nursing Records Using Structured Clinical Knowledge.

Half of nurses' overtime hours are due to records. Nursing records, which are mainly narrative records, cost a large amount of money. However, it has been pointed out that there are problems with their quality and post-use. In this study, we analyzed the value of nursing records for physicians. As a result, we found that the use of standard observation terms in nursing records can create an environment in which patients' conditions can be shared. To create this environment, the physicians of the clinical path committee classified hospitalized patients in terms of disease, treatment, and examination, and created a list of 778 process paths. Physicians, nurses, and researchers collaborated to develop digital contents with high-priority observation items and care actions adapted to patient conditions for each path. We developed a clinical support system equipped with these digital contents. In May 2019, we installed the system in a 900-bed university hospital. Then, in October 2020, we installed the system in a 400-bed general hospital. We used "nurses' overtime hours for recording" and "reduction rate" as indicators of the usefulness of this system. In the 900-bed university hospital, we compared the previous year's results for March, the end of the fiscal year. This overtime hours were 2,944 hours 00 minutes in March 2019 and 2,141 hours 55 minutes in March 2020. 27% reduction was indicated. The respective bed occupancy rates were 90.80 percent and 90.60 percent, with no difference. In the 400-bed general hospital, This overtime hours were compared to the previous year, covering November and December after one month of implementation. 386 hours in November 2019 and 204.5 hours in November 2020. 47% reduction indicated. 366 hours in December 2019 and 214.5 hours in December 2020. A reduction of 41% was shown. These results suggest that the implementation of this system can both improve the quality of team care and reduce overtime.

Nursing Record Innovations Aimed at Harmonizing Structured Clinical Knowledge Among Doctors and Nurses.

Hospitalization expenses account for a high proportion of national medical care expenditure in Japan. In 2015, the total national medical care expenditure in Japan was 42.4 trillion yen, and hospitalization expenses were 15.6 trillion yen (36.8%). Therefore, it is necessary to reduce hospitalization expenses. The labor cost of physicians and nurses accounted for about 1/3rd of all expenditure of general hospitals in 2015. Moreover, the personnel cost of nurses accounted for about 1/5th of all expenditure, indicating that it has a marked impact on hospital management. Nurses spend a lot of time completing descriptive records; however, the quality of such records is poor. It is necessary to improve nurse's records to make them highly accessible and reduce the amount of time nurses spend producing records. The objective of this study was to improve the processes underlying record-keeping by nurses in order to harmonize structured clinical knowledge among doctors and nurses. We created 778 Patient Condition Adaptive Path System (PCAPS) items, covering all of the clinical departments that were registered for the PCAPS content master. The resultant masters will be standardized by sharing them with hospitals that adopt the "Team Compass" application. We were able to summarize all of the information in clinical progress sheets because we could link the information described in electronic medical records with that described in Team Compass. Therefore, it became easy to collect information by linking information about clinical orders. The system also made it possible for foundational nursing plans to be created in collaboration with doctors instead of being developed by nurses alone because it allowed information regarding patients' problems, the clinical process, and observation selection to be shared smoothly with doctors. We implemented Team Compass in May 2019. On the first day, PCAPS-based care pathways were used to treat 580 of 623 inpatients. Approximately 4,000 patients were treated using this system from May to August 2019. No major problems have arisen since the implementation of Team Compass.

Comparison of time to positivity of pediatric blood cultures obtained within the first year of life and in later years.

The time to positivity (TTP) of blood culture has significant value for clinicians. However, almost all subjects of previous studies regarding TTP were adults and early infants. Therefore, careful attention is required when referring to previously published data, which might differ according to the age of subjects, as the tendency of infectious focus and pathogens identified from culture vary with age. In this study, we compared the TTP between two pediatric age groups (≤12 months and 13 months to 15 years [>12 months]) at a teaching hospital during a 5-year period. Of the 95 subjects, 41 and 54 were aged ≤12 and > 12 months, among whom true pathogenic bacteria were identified in 12 (29.3%) and 19 (35.2%), respectively. The median TTP for the younger group with pathogenic bacteria was 11.2 (interquartile range, 10.0-11.9) hours, which was significantly shorter than that for the older group (12.6 [interquartile range, 11.9-16.9] hours) (P = 0.01). At 12 h after the initiation of culture, the younger group with pathogenic bacteria had a significantly higher positivity rate (83.3%) than the older group (26.3%) (P < 0.01). The times required for the positivity to exceed 90% were 13.4 and 20.1 h for the younger and older pathogenic groups and 30.4 and 67.8 h for the younger and older contaminant groups, respectively. The range of TTP could be assessed more accurately by considering the effect of age on the infectious background.

Reduction of Overwork Time of Nurses by Innovation of Nursing Records Using Structured Clinical Knowledge.

Hospitalization expenses account for a high rate of national medical care expenditure in Japan. The Japanese national medical care expenditure was 42 trillion 364.4 billion yen in 2015, in which hospitalization expenses were 15 trillion 575.2 billion yen (36.8%). Therefore, it is necessary to take measures to reduce hospitalization expenses. The total ratio of the labor cost of physicians and nurses accounted for about 1/3 of all expenditures of general hospitals in 2015. Moreover, the personnel cost of nurses accounted for about 1/5 of all expenditure, showing that the personnel cost of nurses is an element with a large influence on hospital management. The objective of this study was to develop a methodology to reduce the overtime work of nurses accounting for a large rate of personnel expenses by focusing on overtime work, a personnel expense-increasing factor, aiming at hospital cost reduction. First, the cause of overtime work, planning, and recording by nurses were analyzed and an IT application increasing the quality and efficiency of the work was developed. Then, fees for the use and maintenance of the IT system meeting the following conditions were set as a strategy to introduce the system: (1) 50% reduction of the overtime work of nurses and (2) fees 50% or lower than the reduced payment for overtime work. This IT application was introduced to the heads and directors of nursing of 5 hospitals and the strategy was proposed. All heads and directors highly evaluated the system and responded to initiate the process for the introduction. It was suggested that the methodology to reduce the overtime work of nurses proposed by this study is useful and feasible.

Amoxicillin effect on bacterial load in group A streptococcal pharyngitis: comparison of single and multiple daily dosage regimens.

BACKGROUND: Culture tests have demonstrated that once-daily administration of amoxicillin may be effective in the treatment of group A streptococcal (GAS) pharyngitis. However, culture methods do not allow accurate assessments of bacterial load changes because of the suppressive effect of the antibiotic on bacterial growth. In this study, we used real-time PCR to compare the effectiveness of once-daily and multiple-daily amoxicillin treatment for pediatric patients with GAS pharyngitis. METHODS: The subjects were children (≧3 years of age) diagnosed with GAS pharyngitis. Amoxicillin was administered at a dose of 40-50 mg/kg/day, divided into one (QD), two (BID), or three (TID) daily doses, for 10 days. Throat swabs were collected before treatment (visit 1), 1 to 3 days after treatment (visit 2), and 9 to 11 days after treatment (visit 3), and GAS copies were quantified by real-time PCR. The main compared parameters were the rate of negative PCR results and the number of GAS determined by PCR in throat swabs between each regimen. RESULTS: Samples were collected from 34 patients (QD, 12; BID, 15; TID, 7) at visit 1, 32 patients (QD, 11; BID, 14; TID, 7) at visit 2, and 25 patients (QD, 7; BID, 11; TID, 7) at visit 3. The rates of negative PCR result for QD, BID, and TID regimens were 18.2, 0, and 14.3% at visit 2, and 85.7, 72.7, and 85.7% at visit 3, respectively. The median values of bacterial load for QD, BID, and TID groups at visit 1 were 1.4 × 106, 8.2 × 105, and 5.4 × 105 copies/µL. At visit 2, they comprised 3.8 × 103, 1.1 × 103, and 2.8 × 103 copies/µL, respectively, whereas at visit 3, GAS copies were mostly undetectable. There was no statistical difference in the negative results and median value of GAS copies between regimens at any stage. CONCLUSIONS: Our results obtained by a molecular biology approach indicated that the QD regimen was as effective in eradicating GAS infection as BID or TID. TRIAL REGISTRATION: UMIN000036083 / March 12, 2019.

Urinary angiotensinogen in pediatric urinary tract infection.

BACKGROUND: Urinary tract infection (UTI) is one of the most common diseases in children, and urinary angiotensinogen (U-AGT) is a new biomarker gathering attention in many renal diseases. U-AGT reflects intrarenal renin-angiotensin system (RAS) activity. We conducted a study to measure U-AGT in children <4 months old with UTI. METHODS: All children <4 months old who came to Toshima Hospital with fever between January 2015 and December 2015 were included. Patients were divided into a UTI group and a non-UTI group, and U-AGT was measured. RESULTS: Median U-AGT was higher in patients with UTI compared with patients without UTI: (0.56 ng/dL, range, 0.025-2.753 ng/dL vs 0.13 ng/dL, range, 0.008-1.697 ng/dL, respectively; P < 0.05). CONCLUSIONS: U-AGT is elevated in UTI patients, and RAS activation may contribute to renal injury caused by UTI.

Intestinal carriage of methicillin-resistant Staphylococcus aureus in nasal MRSA carriers hospitalized in the neonatal intensive care unit.

BACKGROUND: The current data regarding the correlation between the methicillin-resistant Staphylococcus aureus (MRSA) clones carried in the nasal cavity and digestive tract are inadequate. METHODS: MRSA strains were isolated from both the feces and nasal swabs of 21 nasal-MRSA carriers ranging from 10 to 104 days of age treated at the neonatal intensive care units of two hospitals. The molecular epidemiological characteristics of the isolates were determined: multilocus sequence types, spa-types, staphylococcal cassette chromosome mec (SCCmec) types, carriage of four exotoxin genes, and genes contained in commercially available kit. RESULTS: The feces of all nasal carriers contained MRSA at levels ranging from 4.0 × 10(2) to 2.8 × 10(8) colony forming units/g feces. The MRSA clones isolated from the feces and the nasal swabs of each patient were the same. Four MRSA clones, clonal complex (CC) 8-SCCmec IVl, CC8-SCCmec IVb, CC1-SCCmec IVa and CC5-SCCmec IIa were identified from 21 patients. All CC8-SCCmec IVl strains and one of three CC5-SCCmec IIa strains carried the toxic shock syndrome toxin gene. CONCLUSIONS: The feces of tested MRSA carriers contained the same MRSA clones as the nasal isolates in considerable amounts, suggesting that more careful attention should be paid for the handling of excrement in the case of newborn babies or infants than that of adults.

The clinical utility of a near patient care rapid microarray-based diagnostic test for influenza and respiratory syncytial virus infections in the pediatric setting.

We evaluated the potential clinical utility of an automated near patient molecular assay Verigene Respiratory Virus Plus (RV+) and rapid immunochromatographic antigen tests (RIAT) in the pediatric setting for diagnosis of influenza and respiratory syncytial virus infections when testing was performed by the pediatrician seeing the patient. Overall, with respect to influenza virus, sensitivity and specificity for RIAT were 70.8% and 100%, respectively, compared to 100% and 96.2%, respectively, for RV+. For respiratory syncytial virus, sensitivity and specificity for RIAT were 78.9% and 100%, respectively, compared to 100% and 100%, respectively, for RV+. When RIAT and RV+ sensitivity for influenza virus was compared based on the time the patient presented after onset of fever, the sensitivity of RIAT at 6 hours was 37.5% compared to 100% for RV+. At 12 hours, RIAT improved to 60.9%. This study confirms the clinical utility of RV+ in the pediatric setting.

snOPY: a small nucleolar RNA orthological gene database.

BACKGROUND: Small nucleolar RNAs (snoRNAs) are a class of non-coding RNAs that guide the modification of specific nucleotides in ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). Although most non-coding RNAs undergo post-transcriptional modifications prior to maturation, the functional significance of these modifications remains unknown. Here, we introduce the snoRNA orthological gene database (snOPY) as a tool for studying RNA modifications. FINDINGS: snOPY provides comprehensive information about snoRNAs, snoRNA gene loci, and target RNAs. It also contains data for orthologues from various species, which enables users to analyze the evolution of snoRNA genes. In total, 13,770 snoRNA genes, 10,345 snoRNA gene loci, and 133 target RNAs have been registered. Users can search and access the data efficiently using a simple web interface with a series of internal links. snOPY is freely available on the web at http://snoopy.med.miyazaki-u.ac.jp. CONCLUSIONS: snOPY is the database that provides information about the small nucleolar RNAs and their orthologues. It will help users to study RNA modifications and snoRNA gene evolution.

On cooperative and efficient overlay network evolution based on a group selection pattern.

In overlay networks, the interplay between network structure and dynamics remains largely unexplored. In this paper, we study dynamic coevolution between individual rational strategies (cooperative or defect) and the overlay network structure, that is, the interaction between peer's local rational behaviors and the emergence of the whole network structure. We propose an evolutionary game theory (EGT)-based overlay topology evolution scheme to drive a given overlay into the small-world structure (high global network efficiency and average clustering coefficient). Our contributions are the following threefold: From the viewpoint of peers' local interactions, we explicitly consider the peer's rational behavior and introduce a link-formation game to characterize the social dilemma of forming links in an overlay network. Furthermore, in the evolutionary link-formation phase, we adopt a simple economic process: Each peer keeps one link to a cooperative neighbor in its neighborhood, which can slightly speed up the convergence of cooperation and increase network efficiency; from the viewpoint of the whole network structure, our simulation results show that the EGT-based scheme can drive an arbitrary overlay network into a fully cooperative and efficient small-world structure. Moreover, we compare our scheme with a search-based economic model of network formation and illustrate that our scheme can achieve the experimental and analytical results in the latter model. In addition, we also graphically illustrate the final overlay network structure; finally, based on the group selection model and evolutionary set theory, we theoretically obtain the approximate threshold of cost and draw the conclusion that the small value of the average degree and the large number of the total peers in an overlay network facilitate the evolution of cooperation.

On cooperative and efficient overlay network evolution based on a group selection pattern.

In overlay networks, the interplay between network structure and dynamics remains largely unexplored. In this paper, we study dynamic coevolution between individual rational strategies (cooperative or defect) and the overlay network structure, that is, the interaction between peer's local rational behaviors and the emergence of the whole network structure. We propose an evolutionary game theory (EGT)-based overlay topology evolution scheme to drive a given overlay into the small-world structure (high global network efficiency and average clustering coefficient). Our contributions are the following threefold: From the viewpoint of peers' local interactions, we explicitly consider the peer's rational behavior and introduce a link-formation game to characterize the social dilemma of forming links in an overlay network. Furthermore, in the evolutionary link-formation phase, we adopt a simple economic process: Each peer keeps one link to a cooperative neighbor in its neighborhood, which can slightly speed up the convergence of cooperation and increase network efficiency; from the viewpoint of the whole network structure, our simulation results show that the EGT-based scheme can drive an arbitrary overlay network into a fully cooperative and efficient small-world structure. Moreover, we compare our scheme with a search-based economic model of network formation and illustrate that our scheme can achieve the experimental and analytical results in the latter model. In addition, we also graphically illustrate the final overlay network structure; finally, based on the group selection model and evolutionary set theory, we theoretically obtain the approximate threshold of cost and draw the conclusion that the small value of the average degree and the large number of the total peers in an overlay network facilitate the evolution of cooperation.

Characteristics of ClC7 Cl- channels and their inhibition in mutant (G215R) associated with autosomal dominant osteopetrosis type II in native osteoclasts and hClcn7 gene-expressing cells.

ClC7 Cl(-) channels (Clcn7) are crucial for osteoclastic bone resorption and have heterozygous mutation in autosomal osteopetrosis type II (ADO II) patients. Although extracellular acidification is known to induce ClC7 Cl(-) currents in Clcn7-transfected oocytes, other characteristics of this acid-induced Cl(-) current, as well as the effects of mutant Clcn7 in ADO II, remain to be determined. The present study showed that extracellular acidification evoked outward Cl(-) currents in mouse osteoclasts. Expression of wild-type human Clcn7 in HEK293 cells also induced a significant increase in acid-activated Cl(-) currents. These acid-activated Cl(-) currents were independent of intracellular acidification and [Ca(2+)]( i ) increase. HEK293 cells with the Clcn7 mutation associated with ADO II at G215R did not display these Cl(-) currents. These results suggest that osteoclastic ClC7 Cl(-) channels are activated under extracellar acidification and suppressed in Clcn7 mutant associated with ADO II during bone resorption.

The association of Notch2 and NF-kappaB accelerates RANKL-induced osteoclastogenesis.

Notch signaling plays a key role in various cell differentiation processes including bone homeostasis. However, the specific involvement of Notch in regulating osteoclastogenesis is still controversial. In the present study, we show that RANKL induces expression of Jagged1 and Notch2 in bone marrow macrophages during osteoclast differentiation. Suppression of Notch signaling by a selective gamma-secretase inhibitor or Notch2 short hairpin RNA suppresses RANKL-induced osteoclastogenesis. In contrast, induction of Notch signaling by Jagged1 or by ectopic expression of intracellular Notch2 enhances NFATc1 promoter activity and expression and promotes osteoclastogenesis. Finally, we found that Notch2 and p65 interact in the nuclei of RANKL-stimulated cells and that both proteins are recruited to the NFATc1 promoter, driving its expression. Taken together, our results show a new molecular cross talk between Notch and NF-kappaB pathways that is relevant in osteoclastogenesis.

RANKL-stimulated TNFalpha production in osteoclast precursor cells promotes osteoclastogenesis by modulating RANK signaling pathways.

Although TNFalpha is known to be an important factor for bone resorption, particularly in inflammatory bone diseases, the relevance between RANKL and TNFalpha in osteoclastogenesis remains unclear. In this study we examined the mechanism of TNFalpha induced osteoclastogenesis and its downstream signaling. We show that osteoclastogenesis is suppressed by anti-TNFalpha- and anti-TNF receptor type I (TNFRI)-antibodies and in TNFalpha- and TNFRI-deficient mice using in vitro culture systems: (1) co-culture of mouse spleen derived osteoclast precursor cells (pOCs) with osteoblasts, (2) pure pOC culture and (3) RAW264.7 cells in presence of RANKL. Furthermore, TNFalpha production in pOCs was stimulated by RANKL. Endogenous TNFalpha in pOCs induced c-Fos and NFATc1. Expression rates of NFATc1 and c-Fos were significantly decreased in TNFalpha- and TNFRI-deficient pOCs during osteoclastogenesis. These results indicate that TNFalpha is induced by RANKL in pOCs and serves as an autocrine factor promoting osteoclastogenesis through c-Fos and NFATc1 activation.

Three Na+/Ca2+ exchanger (NCX) variants are expressed in mouse osteoclasts and mediate calcium transport during bone resorption.

The plasma membrane Na(+)/Ca(2+) exchanger (NCX) is a bidirectional transporter that mediates the exchange of Na(+) for Ca(2+) depending on the electrochemical gradients. Mammalian NCXs form a multigene family comprising NCX1, NCX2, and NCX3 isoforms. Although it has been known that NCX1 in rat osteoclasts is coupled with the Na(+)/ H(+) exchanger for regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)), it is unclear what kind of NCX1 variants are expressed and whether the other two NCX isoforms are also present in mouse osteoclasts. To clarify the role of NCXs during bone resorption, we investigated the expression of NCXs, the ion transport via NCXs, and the effects of NCX inhibitors on bone-resorbing activity in mouse osteoclasts. Using RT-PCR, immunocytochemical, and Western blot methods, we detected three splice variants of NCX1 and NCX3, namely NCX1.3, NCX1.41, and NCX3.2. Of these, NCX1.41 is a newly identified splice variant. Low extracellular sodium ([Na(+)](o)) solution increases the intracellular Ca(2+) concentration via NCX transporter in fura-2-loaded osteoclasts. The [Na(+)](o)-free solution-induced [Ca(2+)](i) increase was suppressed by benzyloxyphenyl NCX inhibitors. Bidirectional NCX currents in mouse osteoclasts were recorded using the patch clamp method and could be suppressed with NCX inhibitors. NCX inhibitors also decreased the resorption pit area surrounding osteoclasts in a dose-dependent manner. Furthermore, small interference RNAs targeted against NCX1.3, NCX1.41, and NCX3.2 expressed in mouse osteoclasts suppressed osteoclastic pit formation. These results show that three NCX variants are expressed in mouse osteoclasts and play an important role for Ca(2+) transport and regulation during osteoclastic bone resorption.

Ribosomal protein gene knockdown causes developmental defects in zebrafish.

The ribosomal proteins (RPs) form the majority of cellular proteins and are mandatory for cellular growth. RP genes have been linked, either directly or indirectly, to various diseases in humans. Mutations in RP genes are also associated with tissue-specific phenotypes, suggesting a possible role in organ development during early embryogenesis. However, it is not yet known how mutations in a particular RP gene result in specific cellular changes, or how RP genes might contribute to human diseases. The development of animal models with defects in RP genes will be essential for studying these questions. In this study, we knocked down 21 RP genes in zebrafish by using morpholino antisense oligos to inhibit their translation. Of these 21, knockdown of 19 RPs resulted in the development of morphants with obvious deformities. Although mutations in RP genes, like other housekeeping genes, would be expected to result in nonspecific developmental defects with widespread phenotypes, we found that knockdown of some RP genes resulted in phenotypes specific to each gene, with varying degrees of abnormality in the brain, body trunk, eyes, and ears at about 25 hours post fertilization. We focused further on the organogenesis of the brain. Each knocked-down gene that affected the morphogenesis of the brain produced a different pattern of abnormality. Among the 7 RP genes whose knockdown produced severe brain phenotypes, 3 human orthologs are located within chromosomal regions that have been linked to brain-associated diseases, suggesting a possible involvement of RP genes in brain or neurological diseases. The RP gene knockdown system developed in this study could be a powerful tool for studying the roles of ribosomes in human diseases.

Expression of mouse osteoclast K-Cl Co-transporter-1 and its role during bone resorption.

UNLABELLED: To assess the role of Cl- transport during osteoclastic bone resorption, we studied the expression and function of K+/Cl- co-transporters (KCCs). KCC1 and chloride channel-7 were found to be expressed in mouse osteoclasts. The KCC inhibitor, R(+)-butylindazone (DIOA), KCC1 antisense oligo-nucleotides, and siRNA suppressed osteoclastic pit formation. DIOA also decreased Cl- extrusion and reduced H+ extrusion activity. These results show that KCC1 provides a Cl- extrusion mechanism accompanying the H+ extrusion during bone resorption. INTRODUCTION: Mice with deficient chloride (Cl-) channels, ClC7, show severe osteopetrosis, resulting from impairment of Cl- extrusion during osteoclastic bone resorption. However, the expression and functional role of Cl- transporters other than ClC7 in mammalian osteoclasts is unknown. The aim of this study was to determine expression of K+/Cl- co-transporters (KCCs) and their functional role for bone resorption in mouse osteoclasts. MATERIALS AND METHODS: Mouse osteoclasts were derived from cultured bone marrow cells with macrophage-colony stimulating factor (M-CSF) and RANKL or from co-culture of bone marrow cells and primary osteoblasts. We examined the expression of Cl- transporters using RT-PCR, immunochemical, and Western blot methods. The effects of Cl- transport inhibitors on H+ and Cl- extrusion were assessed by measuring intracellular H+ ([H+]i) and Cl- ([Cl-]i). The effects of inhibitors, antisense oligo-nucleotides, and siRNA for Cl- transporters on bone resorption activities were evaluated using a pit formation assay. RESULTS AND CONCLUSIONS: Mouse osteoclasts express not only ClC7 but also K+/Cl- co-transporter mRNA. The existence of KCC1 in the cell membrane of mouse osteoclasts was confirmed by immunochemical staining and Western blot analysis. KCC inhibitors and Cl- channels blockers increased [Cl-]i and [H+]i in resorbing osteoclasts, suggesting that the suppression of Cl- extrusion through KCC and Cl- channels leads to reduced H+ extrusion activity. The combination of both inhibitors greatly suppressed these extrusion activities. KCC inhibitors and Cl- channel blockers also decreased osteoclastic bone resorption in our pit area essay. Furthermore, KCC1 antisense oligo-nucleotides and siRNA suppressed osteoclastic pit formation as well as treatment of ClC7 inhibitors. These results indicate that K+/Cl- co-transporter-1 expressed in mouse osteoclasts acts as a Cl- extruder and plays an important role for H+ extrusion during bone resorption.

Analysis of ribosomal protein gene structures: implications for intron evolution.

Many spliceosomal introns exist in the eukaryotic nuclear genome. Despite much research, the evolution of spliceosomal introns remains poorly understood. In this paper, we tried to gain insights into intron evolution from a novel perspective by comparing the gene structures of cytoplasmic ribosomal proteins (CRPs) and mitochondrial ribosomal proteins (MRPs), which are held to be of archaeal and bacterial origin, respectively. We analyzed 25 homologous pairs of CRP and MRP genes that together had a total of 527 intron positions. We found that all 12 of the intron positions shared by CRP and MRP genes resulted from parallel intron gains and none could be considered to be "conserved," i.e., descendants of the same ancestor. This was supported further by the high frequency of proto-splice sites at these shared positions; proto-splice sites are proposed to be sites for intron insertion. Although we could not definitively disprove that spliceosomal introns were already present in the last universal common ancestor, our results lend more support to the idea that introns were gained late. At least, our results show that MRP genes were intronless at the time of endosymbiosis. The parallel intron gains between CRP and MRP genes accounted for 2.3% of total intron positions, which should provide a reliable estimate for future inferences of intron evolution.