The ART of mating: alternative reproductive tactics and mating success in a nest-guarding fish.

Behavioural observations in the field of male Mediterranean damselfish Chromis chromis were combined with molecular analyses, using bi-parentally and maternally inherited markers, to investigate reproductive success patterns of alternative reproductive tactics (ARTs) in terms of number of eggs sired and number of females contributing to each nest. Cuckoldry was observed in every nest sampled, with at least two and up to seven sneaker males per nest. The nesting male, however, always significantly fertilized the greater number of eggs (on average 49%) in each clutch, whereas each sneaker fertilized around 7% of the clutch. The average number of females whose eggs were fertilized by nesting males was 6·76 (range 2-13), while each sneaker on average fertilized the eggs of 1·74 (range 1-8) females. Using this sibship reconstruction, some of the factors involved in the regulation of the dynamic equilibrium of reproductive success were investigated between the two ARTs shown by C. chromis males. Results show that the sneakers' reproductive success was positively linked to egg clutch size; the density of individuals in the nesting area negatively affected the size of egg clutches; the rate of defence behaviours performed by nesting males negatively influenced the number of females contributing to each nest.

Parental education and youth suicidal behaviours: a systematic review and meta-analysis.

AIMS: Lower parental education has been linked to adverse youth mental health outcomes. However, the relationship between parental education and youth suicidal behaviours remains unclear. We explored the association between parental education and youth suicidal ideation and attempts, and examined whether sociocultural contexts moderate such associations. METHODS: We conducted a systematic review and meta-analysis with a systematic literature search in PubMed, PsycINFO, Medline and Embase from 1900 to December 2020 for studies with participants aged 0-18, and provided quantitative data on the association between parental education and youth suicidal ideation and attempts (death included). Only articles published in English in peer-reviewed journals were considered. Two authors independently assessed eligibility of the articles. One author extracted data [e.g. number of cases and non-cases in each parental education level, effect sizes in forms of odds ratios (ORs) or beta coefficients]. We then calculated pooled ORs using a random-effects model and used moderator analysis to investigate heterogeneity. RESULTS: We included a total of 59 articles (63 study samples, totalling 2 738 374 subjects) in the meta-analysis. Lower parental education was associated with youth suicidal attempts [OR = 1.12, 95% Confidence Interval (CI) = 1.04-1.21] but not with suicidal ideation (OR = 1.05, 95% CI = 0.98-1.12). Geographical region and country income level moderated the associations. Lower parental education was associated with an increased risk of youth suicidal attempts in Northern America (OR = 1.26, 95% CI = 1.10-1.45), but with a decreased risk in Eastern and South-Eastern Asia (OR = 0.72, 95% CI = 0.54-0.96). An association of lower parental education and increased risk of youth suicidal ideation was present in high- income countries (HICs) (OR = 1.14, 95% CI = 1.05-1.25), and absent in low- and middle-income countries (LMICs) (OR = 0.91, 95% CI = 0.77-1.08). CONCLUSIONS: The association between youth suicidal behaviours and parental education seems to differ across geographical and economical contexts, suggesting that cultural, psychosocial or biological factors may play a role in explaining this association. Although there was high heterogeneity in the studies reviewed, this evidence suggests that the role of familial sociodemographic characteristics in youth suicidality may not be universal. This highlights the need to consider cultural, as well as familial factors in the clinical assessment and management of youth's suicidal behaviours in our increasingly multicultural societies, as well as in developing prevention and intervention strategies for youth suicide.

Self-reported sleep disorders in secondary school students: an epidemiological and risk behavioural analysis.

PURPOSE: To estimate the prevalence of self-reported sleep disorders (SD), to examine associations among demographic characteristics and familiar factors with SD, between SD and daytime sleep-related disorders (DD) and between evening habits and SD. METHODS: An anonymous questionnaire was proposed to 1563 students (aged 14-21 years, mean age 16.5 +/- 1.5; 42.8% males, 57.2% females) attending all classes of two high schools in Verona (North-East of Italy). Data were analyzed by some personal and familial characteristics, by definition of three sleeper groups (non problem, occasional problem or problem-sleepers). Moreover SD were put in relation with DD and with some personal evening attitudes. RESULTS: The 75.5% of the subjects report at least one SD. Difficulty falling asleep is the most frequent SD. The DD concern 91.2% of the sample. Females are more involved than males in SD and DD. All SD result strongly associated with the referred DD, except for sleepiness. Sport is significantly correlated with a minor prevalence of SD. Smoking and studying appear to be associated with SD. CONCLUSIONS: Since SD in youth constitute an important Public Health matter with a severe social impact they would be accurately studied to offer youth appropriate counselling given the importance of lifestyle in determining good sleep.

Randomized controlled trial of duration of analgesia following intravenous or rectal acetaminophen after adenotonsillectomy in children.

BACKGROUND: Doses of acetaminophen 40 mg kg(-1) rectally and 15 mg kg(-1) i.v. produce similar effect-site concentrations. However, the clinical effectiveness of these routes has not been compared. The aim of this study was to compare duration and efficacy of analgesia in children following adenotonsillectomy after acetaminophen either 40 mg kg(-1) rectally or 15 mg kg(-1) i.v. METHODS: Fifty children aged between 2 and 5 yr were recruited. They received a standardized anaesthetic, including 2 microg kg(-1) of fentanyl. Children were randomized to receive either rectal or i.v. acetaminophen. Postoperative pain was assessed regularly with the Children and Infants Postoperative Pain Scale score and rescue analgesia provided if scores were 4 or greater. The primary outcome measure was time to first analgesia. Results were plotted with a Kaplan-Meier analysis and median time to rescue analgesia compared between the groups. RESULTS: The protocol was successfully completed in 46 children. Forty-five children required rescue medication. The time to first rescue analgesia was longer in children receiving rectal acetaminophen (median 10 h, inter-quartile range 9-11 h) compared with those receiving i.v. acetaminophen (7, 6-10 h) with a P-value of 0.01 by log-rank test for equality in survivor function. Few children in either group required rescue analgesia within the first 6 h with differences between the groups being most prominent in the period from 6 to 10 h. CONCLUSIONS: Rectal acetaminophen 40 mg kg(-1) provides longer analgesia for moderately painful procedures when compared with 15 mg kg(-1) acetaminophen i.v.

Does infant negative emotionality moderate the effect of maternal depression on motor development?

Maternal depression represents an important social/environmental factor in early childhood; however, its effect on children's motor development may vary depending on the role of infants' dispositional variables. The objective of this study is to investigate the effect of the interaction between maternal depressive symptoms in the first two years of a child's life and the child's temperamental negative emotionality on motor development during this time. Using a cross-sectional study, we assessed 272 infants aged 0 to 24 months old and their mothers. We measured the following variables: maternal depression, infant's negative emotionality, and motor development. A three-way interaction effect highlights that negative emotionality in infants and maternal depression together affect children's overall motor growth trajectory. Infants with low negative emotionality display no effect of maternal depression on motor development. Conversely, infants with high negative emotionality seem to be more susceptible to the effect of maternal depression. Specifically, high maternal depression tends to foster the negative effect of infant's negativity on motor development across time, albeit not significantly. Finally, the absence of maternal depression significantly buffers negative temperament in infants. Findings highlighted the importance of integrating different perspectives when describing early motor growth. In fact, only when considering the interdependence of potential predictors their effect on the motor growth significantly emerges. Screening for early temperamental vulnerability might help in tailoring interventions to prevent maternal depression from affecting infants' motor development.

Italian translation and cross-cultural comparison with the Childhood Attachment and Relational Trauma Screen (CARTS).

Background: The Childhood Attachment and Relational Trauma Screen (CARTS) is a computer-administered survey designed to assess retrospectively the socio-ecological context in which instances of child abuse may have occurred. To date, studies supporting the validity of the CARTS have only been undertaken in English-speaking North American populations. Validation projects in other countries and cross-cultural comparisons are therefore warranted. Objective: Develop and preliminarily evaluate the psychometric properties of an Italian version of the CARTS on college students and compare such observations to data acquired from Canadian students. Method: Seventy-nine undergraduate students from the University of Padua (Italy) completed an Italian translation of the CARTS as well as measures of childhood experiences, mental health and attachment, responses to which were compared to those obtained in 288 Canadian students who completed the CARTS in English. Results: Internal consistency and convergent validity with the Childhood Trauma Questionnaire and Parental Bonding Instrument were found to be acceptable for the Italian translation. Within the Italian sample, correlation analyses suggested that CARTS Mother ratings referring to attachment and abuse were associated with romantic attachment, whereas CARTS Father ratings were significantly correlated to PTSD symptoms and other symptoms of psychopathology-distress. Significant differences between Italian and Canadian students across the relationship types for the CARTS abuse and attachment scales were found, indicating that Italian students rated their mothers and fathers as simultaneously less abusive, but also less as a source of secure attachment. Conclusions: The results of this preliminary study seem to suggest convergent validity of the Italian CARTS and the association between childhood attachment-related experiences and romantic attachment. Cultural variations were identified between Canadian and Italian students in both attachment and abuse scales. Future studies to investigate cross-cultural variations in the relational context of childhood abuse and in order to boost Italian CARTS psychometric features are warranted.

Planteamiento: La Encuesta de Apego Infantil y Trauma Relacional (CARTS) es una encuesta administrada por ordenador diseñada para evaluar retrospectivamente el contexto socio-ecológico en el que pueden haber ocurrido casos de abuso infantil. Hasta la fecha, los estudios que apoyan la validez de la CARTS sólo se han realizado en poblaciones norteamericanas de habla inglesa. Por lo tanto, se justifican los proyectos de validación en otros países y las comparaciones interculturales. Objetivos: Desarrollar y evaluar de manera preliminar las propiedades psicométricas de una versión italiana de CARTS en estudiantes universitarios y comparar dichas observaciones con datos obtenidos de estudiantes canadienses. Método: Setenta y nueve estudiantes de pre-grado de la Universidad de Padua (Italia) completaron una traducción al italiano de la CARTS, así como medidas de experiencias infantiles, salud mental y apego. Las respuestas fueron comparadas con las obtenidas en 288 estudiantes canadienses que completaron la CARTS en inglés. Resultados: Se encontró que la coherencia interna y la validez convergente con el Cuestionario de Trauma Infantil (Childhood Trauma Questionnaire) y el Instrumento de Vinculación Parental (Parental Bonding Instrument) eran aceptables para la traducción al italiano. Dentro de la muestra italiana, los análisis de correlación sugirieron que las puntuaciones de la CARTS-Madre que se refieren al apego y al abuso se asociaron con el apego romántico, mientras que las puntuaciones de la CARTS-Padre se correlacionaron significativamente con síntomas de TEPT y otros síntomas de trastorno psicopatológico. Se encontraron diferencias significativas entre los estudiantes italianos y canadienses entre los tipos de relación para las escalas de abuso y apego de la CARTS, lo que indica que los estudiantes italianos clasificaron a sus madres y padres simultáneamente como menos abusivos, pero también menos como fuente de apego seguro. Conclusiones: Los resultados de este estudio preliminar parecen sugerir la validez convergente de la CARTS italiana y la asociación entre las experiencias relacionadas con el apego infantil y el apego romántico. Se identificaron variaciones culturales entre estudiantes canadienses e italianos en las escalas de apego y abuso. Se justifican la realización de futuros estudios para investigar las variaciones interculturales en el contexto relacional del abuso infantil y con el fin de impulsar las características psicométricas de la CARTS italiana.

Detection and genetic characterization of Porcine circovirus type 3 in Italy.

Porcine circovirus type 3 (PCV3) was recently proposed as a new porcine circovirus. It has been described by researchers in the USA and China and associated with porcine dermatitis and nephropathy syndrome, reproductive failure and systemic inflammation disease. The study reports the occurrence of the new virus in Italy. PCV3 was detected in the tissues of foetuses and stillborn piglets coming from two farms located in the Po Valley. The genome sequences of the two Italian strains share 99.7% to 97.8% of nucleotide identity with those available in GenBank. Results strengthen the hypothesis of PCV3 as a new emerging porcine circovirus, widespread all over the world. It follows the urgency of investigating in depth epidemiology and pathogenicity associated with this new virus.

Performance of nested RT-PCR on CSF for tuberculous meningitis diagnosis in HIV-infected patients.

SETTING: Timely diagnosis of tuberculous meningitis (TBM) in patients with human immunodeficiency virus (HIV) infection remains a challenge. Despite the current scale-up of the Xpert® MTB/RIF assay, other molecular diagnostic tools are necessary, particularly in referral centres in low- and middle-income countries without Xpert testing. OBJECTIVE: To determine the diagnostic performance of nested real-time polymerase chain reaction (nRT-PCR) in HIV-infected TBM patients categorised according to standardised clinical case definitions. DESIGN: Based on clinical, laboratory and imaging data, HIV-infected patients with suspected TBM were prospectively categorised as 'definite TBM', 'probable TBM', 'possible TBM' or 'not TBM'. We evaluated nRT-PCR sensitivity and specificity in diagnosing TBM among definite TBM cases, and among definite + probable TBM cases. RESULTS: Ninety-two participants were enrolled in the study. nRT-PCR sensitivity for definite TBM (n = 8) was 100% (95%CI 67-100) and 86% (95%CI 60-96) for both definite and probable TBM (n = 6). Assuming that 'not TBM' patients (n = 74) were true-negatives, nRT-PCR specificity was 100% (95%CI 95-100). The possible TBM group (n = 4) had no nRT-PCR positives. CONCLUSIONS: The nRT-PCR is a useful rule-in test for HIV-infected patients with TBM according to international consensus case definitions. As nRT-PCR cannot exclude TBM, studies comparing and combining nRT-PCR with other assays are necessary for a rule-out test.

Inhibition of cell proliferation by the interferon-inducible 204 gene, a member of the Ifi 200 cluster.

The role of the IFN-inducible p204 as growth regulator was investigated by transfecting an expression vector constitutively expressing p204 into several cell lines. Like pRB and p107, p204 is a potent growth inhibitor in sensitive cells, as demonstrated by the cell focus assay. Since stable transfectants of sensitive lines constitutively overexpressing p204 could not be established in vitro, we inserted the 204 cDNA into a vector bearing an heavy-metal-inducible promoter. Here we show that proliferation of B6MEF fibroblasts lacking endogenous p204 is strongly inhibited by transient p204 expression in the nucleus. p204 delays G1 progression into the S-phase and cells accumulate with a DNA content equivalent to cells arrested in late G1. Moreover, the role of p204 in the control of cell growth in vivo was investigated by generating transgenic mice in which the Ifi 204 gene was constitutively expressed in all tissues. To this end, expression vectors bearing the 204 cDNA under the control of the SV40 viral promoter were constructed. The overexpression of the p204 transgene achieved by injecting fertilized mouse eggs with these vectors was compatible with embryo development up to the four-cell stage in an in vitro follow-up of 4.5 days. However, no viable animals with an intact copy of the transgene were obtained, suggesting that high and constitutive levels of p204 expression can impair normal embryo development. These findings indicate that p204 plays a negative role in growth regulation and provide new information about the molecular mechanisms exploited by IFNs to inhibit cell proliferation.

Shielding properties of laser-induced plasmas in ocular media irradiated by single Nd:YAG pulses of different durations.

Shielding of laser pulses by plasmas generated in ocular media has been experimentally investigated using single Nd:YAG laser pulses of different durations, ranging from several nanoseconds (ns) to a few tens of picoseconds (ps), both in saline solution and extracted calf vitreous. At the respective threshold radiant exposures for breakdown in saline (259 mJ/cm2 for 7 ns, 20.5 mJ/cm2 for 220 ps and 4.7 mJ/cm2 for 30 ps pulses) the single pulse energy transmission is found to be about 74% for 7 ns, 55% for 220 ps and 50% for 30 ps, thus showing that shielding of laser-induced plasmas is more effective for shorter pulses than for longer pulses. Moreover, the decrease in transmission with increasing radiant exposure is faster in the picosecond case than in the nanosecond case. These results show that direct irradiation of the retina is, to some extent, present especially near threshold. In the case of picosecond pulses, a comparison with published results indicates that shielding between subsequent pulses in a train is likely to occur only to a very limited extent.

Antibody studies in mice of outer membrane antigens for use in an improved meningococcal B and C vaccine.

Since 1988, N. meningitidis, B:4:P1.15, ET-5 complex, has been responsible for an epidemic of meningococcal disease in Greater São Paulo, Brazil. Despite current trials to develop an effective vaccine against group B meningococci, children less than 2 years old have not been protected. It has been suggested that iron-regulated proteins (IRPs) should be considered as potential antigens for meningococcal vaccines. The vaccines under study consisted of outer-membrane vesicles depleted of lipooligosaccharide from three serogroup B strains and one serogroup C strain, IRPs, meningococcal group C polysaccharide and aluminum hydroxide. Four different protein and C polysaccharide concentrations were studied. The ELISA and bactericidal results showed a higher antibody response when 2 injections of 2.0 micrograms doses were administered. Despite higher IgG reactivity against antigen preparations containing IRPs seen in ELISA, the bactericidal activity was not increased if the target strain was grown in iron-restricted medium. The influence of addition of alkaline-detoxified lipooligosaccharide (dLOS) on immunogenicity of the vaccine was also investigated, and the dLOS provided for a more functionally specific antibody response.

Peripheral neuropathy associated with experimental plasma cell neoplasm in the mouse.

In order to investigate the role of paraproteins in the production of neuropathy we experimentally induced monoclonal immunoglobulin-producing tumours into 32 BALB/c mice by injecting mineral oil or pristane intraperitoneally. In 11 mice morphologic and histometric studies of the sciatic nerve revealed the presence of neuropathy. Immunohistochemical studies did not demonstrate a significant amount of immunoglobulins and light chains in the endoneurium. The advantages and limits of this experimental model are discussed.

The effect of etoposide (VP-16) on mouse L fibroblasts: modulation of stress response, growth and apoptosis genes.

Molecular events were studied in mouse L cells treated with etoposide (VP-16) a drug widely used in cancer therapy. Modulation of the expression of stress response genes belonging to the hsp70 family (hsp70, hsc73, grp78), growth- and cycle-related genes (c-myc, c-fos, c-jun, histone H3) and apoptosis-related genes (p53, TRPM-2, tTG) was monitored at different time points in the cells that remained adherent to the substrate up to 96 hours after exposure to VP-16. The steady state level of mRNA was determined by Northern blot analysis and hybridization with specific probes, and the relative rate of gene transcription was monitored by run on transcription with isolated nuclei. Our results indicate that protracted VP-16 treatment of 1. cells induces, within 24 hours, the arrest of DNA synthesis, repression of growth-related genes and transient induction of the tTG gene. Altogether these molecular events may contribute to the cytotoxic effect of VP-16. However in cells surviving a longer exposure to the drug, the expression of growth-related genes resumes, even if a blockade in DNA replication persists, and expression of the grp78 gene significantly increases. These data suggest that under continuous treatment with VP-16 a fraction of L cells showing increased resistance to the drug may emerge.

Time-resolved fluorescence spectroscopy of hematoporphyrin-derivative in human lymphocytes.

This paper reports on time-resolved microfluorimetric measurements on hematoporphyrin-derivative (HpD)-treated lymphocytes. HpD is at present widely used as a tumor-locating and photosensitizing drug. It is therefore of great importance to study the extent to which the HpD uptake process depends on cell functional and structural properties. Time-resolved fluorescence measurements in single cells are very useful in this respect, since they give information on the content of fluorescent molecules through fluorescence peak-intensity, and, indirectly, on the binding properties through the fluorescence decay times. In particular, we studied the dependence of HpD fluorescence on the cellular functional state. To this end, we performed in-cell fluorescence measurements on human lymphocytes, both in quiescent conditions and in the pre-replicative phase, after stimulation with phytohemagglutinin (PHA). We found a higher HpD content in stimulated lymphocytes. Moreover, we found a spectral band around 575 nm, corresponding to a particular porphyrin species, in which the differences between normal and stimulated lymphocytes are more striking. The porphyrin species emitting in this band seems to play a role in the specific interaction of HpD with tumors, since a similar emission band has also been found in tumor cells containing HpD.

Spectroscopic studies of hematoporphyrin-derivative in culture medium.

This work reports on studies of hematoporphyrin-derivative (HpD) behaviour in culture medium. Absorption, excitation and emission spectra, together with time-resolved fluorescence measurements, were performed. In previous works, similar studies had been carried out on HpD in saline and in lymphocytes: a new porphyrin species (NPS) and the environmental conditions for its formation in saline were studied. A fluorescent emission similar to that presented by the NPS is reported to be more likely in tumor rather than in normal HpD-treated cells, it was also found in greater amounts in lymphocytes in the pre-replicative phase, as compared with quiescent ones. The higher NPS content in stimulated rather than in quiescent lymphocytes may be due either to a differential uptake, as compared with other HpD components, or to a differential formation rate in cells, because of different microenvironmental conditions. To distinguish between these two main assumptions, the formation of NPS in culture medium was studied. The process was very slow: no NPS appeared within the first 40 h. The incubation time of lymphocytes in culture medium added with HpD in the experiments performed was only 1 h and therefore a differential formation rate of NPS may explain the higher content found in stimulated lymphocytes.

The time-dependent behaviour of hematoporphyrin-derivative in saline: a study of spectral modifications.

Hematoporphyrin-Derivative (HpD), a widely-used tumor-specific photosensitizer, is a complex mixture of porphyrins whose composition has yet to be clarified. This paper reports on the behaviour of HpD in saline. From a spectroscopic point of view, the fresh solution is characterized by two main absorption peaks, attributable to monomeric and dimeric forms. With aging, a new porphyrin species (NPS) appears. To define the NPS, absorption, excitation and emission spectra were measured in different conditions and time-resolved fluorescence measurements were also performed. This species exhibits an absorption/excitation peak at 405 nm, an emission peak at 575 nm and a fluorescence decay time of approximately 3.5 ns. Its formation is strongly influenced by many environmental factors: in particular, gases diluted in the solution, temperature, pH and concentration. The presence of Oxygen and a pH value outside the 6-8 range may be considered inhibiting factors. The NPS seems to be quite important in the understanding of HpD tumor-specificity, since the presence of an emission band similar to the NPS one seems to be favoured in tumor cells as compared with normal cells.

Patterns of intra- and interspecific association of gall-forming sawflies in relation to shoot size on their willow host plant.

Four species of gall-forming sawflies were each frequently found to have clumped distributions among shoots within their willow host plant across four sites and three years. When all species were considered together by clone, year, and site, species showed independence of distribution among shoots two thirds of the time and showed positive covariance one third of the time. When pairs of species were considered separately, but clones were combined within sites and years, 60% of the chi-square tests of association were significant. All but one of the significant tests showed positive associations between pairs of species. The stem galler was positively associated with the leaf folder at all sites in all years, and the petiole galler was positively associated with the stem galler and leaf folder for most year by site combinations. When species paris co-occurred on shoots they were usually found at the same or higher density as when found alone on shoots. Only 2 of 100 tests showed a depressed density of a species when co-occurring on shoots with heterospecifics.All sawfly species were found on shoots that were significantly larger (mean node number) than on shoots without sawflies, and species responded to shoot size variation similarly. Sizes of shoots occupied by heterospecific species combinations were usually significantly larger than shoots with only conspecifics, for all species. These data supported the hypothesis that similar species' responses to within-plant variation would lead to positive rather than negative or random species associations. The data do not support the hypothesis that interspecific competition was important in determining shoot choice or species density.

Variation in herbivore density among host plants and its consequences for community structure : Field studies on willow sawflies.

The densities of four species of gall-forming sawflies were found to vary significantly among willow host plant clones. Two of the speices varied among host plants at four sites in each of three years. The other two species varied in density among host plants at most of the sites in two of the three years. Total sawfly density also varied significantly among clones. Individual species densities on willow clones were significantly positively correlated between years when all sites were combined and frequently when sites were considered separately. Most pairwise species combinations were independent in density between years, but some negative correlations existed between the stem galler and the leaf galler. Gall-former densities also were largely independent among clones within years with all sites combined and with sites considered separately. The significant correlations were nearly all positive. At all four sites the combination of significant variation in sawfly densities among willow clones in the field and independence of species densities among clones resulted in significantly different communities (relative abundance of species) among willow clones in three years. Although sawfly abundances differed substantially among the four sites, this remained true. It is argued that the pattern of community structure among clones is the result of variation in host plant quality of clones. We propose an hypothesis to account for patterns of herbivore species associations based on intrapopulation host plant variation.

Arbitrarily primed-polymerase chain reaction for identification and epidemiologic subtyping of oral isolates of Fusobacterium nucleatum.

BACKGROUND: Fusobacterium nucleatum is the most frequently isolated bacterium in periodontal disease and plays an important role in serious infections in other parts of the body. Arbitrarily primed-polymerase chain reaction (AP-PCR) was used to construct primers for specific identification and subtyping of F. nucleatum. Subtypes may differ in virulence and, hence, are important as periodontal pathogens. Subtypes also may differ in antibiotic susceptibility; therefore, knowing the subtypes may influence choice of treatment. METHODS: We analyzed 70 DNA samples of F. nucleatum isolated from patients with periodontal disease (PD) (N = 32) or AIDS-related PD (N = 8) and from healthy carriers (N = 30). From 90 AP-PCR primers screened, five amplification products were selected, cloned in pCR II vector, and sequenced. These sequences were used to design new pairs of specific primers. Sequences were compared to GenBank entries with BLAST and showed no significant matches. RESULTS: Three primer pairs produced bands of approximately 1 Kb (primer 5059S) or 0.5 Kb (primers FN5047 or M1211) with all F. nucleatum DNAs tested. PCR amplification using primer pair M8171 produced a 1 Kb band with isolates from 7 (22%) PD and 5 (63%) PD-AIDS patients and 9 (30%) healthy controls. Using the same primer pair, 2 other bands of approximately 0.5 Kb and 0.4 Kb were observed with DNA from isolates from 2 (6%) PD and all PD-AIDS patients, but were not observed with DNA samples from healthy controls (P<0.0001). All the primer pairs produced no or different amplicon profiles with DNA samples from bacterial species other than F. nucleatum. CONCLUSIONS: Our results suggest that PCR primer pairs 5059S, FN5047 or M1211 can be used to specifically identify F. nucleatum isolates and distinguish them from other bacteria. The primer pair M8171 could also be used to differentiate F. nucleatum isolated from periodontal patients or healthy individuals. These specific primers can be used in PCR analysis for specific identification of F. nucleatum and to distinguish it from other bacteria associated with human periodontitis. These approaches appear promising in facilitating laboratory identification, molecular subtyping, and taxonomy of putative periodontopathogens.

Cell death induced in L-cells by treatment with thymidine: staging of the process and relationship to apoptosis.

The purpose of this study was to characterize the stages in the development of thymidine-induced cell death. L-cells were characterized by both morphologic and quantitative techniques and evaluated at 24, 48, and 72 h of treatment. Cells first enlarged (stage I); about 50% of these enlarged cells then decreased in size with blebbing and compacting (stage II). This residual cell body transformed into a smooth eosinophilic hyaline body (stage III) by 72 h, many of which could be identified within the vacuolar system of viable cells. These changes were reflected in morphologic counts and Coulter sizing. Cell death (loss of labeled DNA) began in stage II and was most prominent in stage III. No cleavage of DNA into oligonucleosomal fragments was detected by agarose gel electrophoresis at any stage. The similarity of these changes to the complete spectrum of apoptosis in vivo is discussed.