Streptococcus zooepidemicus in dogs: Exploring a canine pathogen through multilocus sequence typing.

Streptococcus equi. subsp. zooepidemicus (S. zooepidemicus) associated diseases in dogs have emerged as a significant concern over recent decades. S. zooepidemicus occurs sporadically in dog populations globally, with increased prevalence in shelters/kennels. This study used multilocus sequence typing (MLST) of 149 independent canine S. zooepidemicus isolates to assess associations between sequence type and breed, country of origin, disease severity, sampling type, year, and behaviour within an outbreak. No clear associations for breed, country, sampling type and year were determined in this study. ST-10 and 123 strains were present within all disease categories, from no clinical signs to severe disease. Assessment of S. zooepidemicus infection in 3 UK outbreaks at the same location found ST-10, 18, 123 strains, and a ST-173 strain in a US outbreak, were associated with haemorrhagic pneumonia and persisted in kennelled populations over time. The ST-173 clonal complex has been noted to have severe virulence capabilities in dogs and other species. S. zooepidemicus seems to thrive in environments with a high risk of transmissibility, overcrowding, stress and naïve populations, particularly for those in shelters/kennels. MLST alone cannot determine the virulence phenotype of S. zooepidemicus in dogs. However, a level of conservancy and diversity within ST allelic loci aids the opportunity to cause severe disease in dogs. Thus, further research into whole genome sequencing and characterising the virulence factors of S. zooepidemicus is warranted in dogs.

Serologic responses correlate with current but not future bacterial shedding in badgers naturally infected with Mycobacterium bovis.

Bovine tuberculosis is a challenging cattle disease with substantial economic costs in affected countries. Eradication in parts of the United Kingdom and Ireland is hindered by transmission of the causative agent Mycobacterium bovis between cattle and European badgers (Meles meles). Diagnostic tests in badgers are of limited accuracy but may help us understand and predict disease progression. This study aimed to determine the practical ability of a commercially available serologic test, the Dual Path Platform VetTB assay (DPP), to predict mycobacterial shedding (i.e. infectiousness) and disease progression in badgers, and whether test outcomes were associated with re-capture. Clinical samples collected from 2014 to 2019 from a wild, naturally infected population of badgers in southwest England were tested using mycobacterial culture (from sputum, urine, faeces, abscesses and bite wounds), an interferon-gamma release assay and the DPP assay. Data were analysed at both individual badger and social group levels using generalised linear and cumulative-link mixed models, and linear regression. Only the highest DPP readings [optical density relative light unit (RLU) levels] were associated with mycobacterial shedding [odds ratio (OR) for DPP levels > 100 RLU in individual badgers: 79.6, 95%CI: 14.7-848; and for social groups: OR: 7.28, 95%CI: 2.94-21.44; compared with levels < 100 RLU]. For individual badgers, RLU levels at first capture were not associated with disease progression at subsequent captures. Finally, badgers with very high DPP levels (> 1000 RLU) were four times less likely to be recaptured (OR: 0.24, 95%CI: 0.07-0.83) than those without a detectable DPP response, which might indicate enhanced mortality. We conclude that DPP levels of > 100 RLU identify badgers that are likely to be shedding M. bovis. Levels of > 1000 RLU identify badgers that are much less likely to be re-captured. These results provide insights into the potential value of existing tests in intervention strategies for managing M. bovis in badgers.

A first-order geochemical budget for suspended sediment discharge to the Bay of Bengal from the Ganges-Brahmaputra river system.

The Ganges-Brahmaputra-Meghna (G-B) river system transports >1 × 109 t/yr of sediment, with an estimated 0.7 × 109 t/yr reaching the Bay of Bengal (BoB). This discharge represents a major input of sediment and associated elements to the global ocean, but quantification of the sediment-element mass reaching the BoB has yet to be fully explored. Published geochemical and suspended sediment data are used to calculate a first-order budget for the modern sediment supply of geochemical elements to the BoB. River profile bulk sediment-element concentrations are calculated based on suspended sediment and element measurements taken in the Ganges and Brahmaputra rivers. A Monte Carlo analysis is applied to account for variable sediment and geochemical contributions from each river. Results show that on average, the G-B system contributes ~5% of the global riverine discharge of solid-phase elements from sediment to the oceans. G-B sediments transport >10% of the global element supply of Hf and Zr. For others, like As and Cu, contributions from the G-B are <5%. Results also show that sediment reaching the BoB is relatively enriched in Hf, Zr, Th, REEs, Sn, and Bi, and majorly depleted in Na and Sr compared to UCC elemental concentrations. While limited by data availability and necessary simplifying assumptions, this study nevertheless provides a reasonable first-order budget for the modern discharge of solid-phase elements to the BoB. Insights from this work are significant for understanding the role of the G-B river system in global elemental cycling, and for providing a basis of comparison for future sediment-element discharge in light of rapid environmental change taking place in the region.

Sensitivity of speleothem records in the Indian Summer Monsoon region to dry season infiltration.

In climates with strongly seasonal rainfall, speleothem-based paleoclimate reconstructions are often thought to reflect wet season conditions, assuming a bias toward the season with greater water supply. This is particularly true in monsoon regions, where speleothem records are interpreted to document monsoon strength changes on multiple timescales. Dry season infiltration variability and rainfall seasonality are not typically considered in these reconstructions, even though cave ventilation could bias speleothem growth toward the cooler season. To investigate the influence of dry season infiltration on speleothem geochemistry, we combine a modern, sub-seasonally resolved trace element record from Mawmluh Cave in Northeast India with forward modeling experiments. We find that variations in the amplitude of seasonal signals in speleothem Mg/Ca, which reflects prior carbonate precipitation, are more sensitive to dry season rather than monsoon season infiltration. This sensitivity may be enhanced by dry season cave ventilation. The Mawmluh speleothem Mg/Ca record is consistent with increased dry season rainfall during the 1976-1998 warm phase of the Pacific Decadal Oscillation relative to 1964-2013. Our work demonstrates the importance of considering non-monsoon season rainfall when interpreting speleothem paleoclimate records and suggests that trace elements could provide insight into periods of enhanced dry season infiltration in monsoonal climates.

Semen quality in men with Y chromosome aberrations.

Infertile males sometimes bear structurally balanced chromosome aberrations, such as translocations and inversions, which involve both autosomes and sex chromosomes. The aim of this study was to evaluate genotype-phenotype correlations in a sample of infertile men with various types of Y chromosome abnormalities. In particular, we examined the effect of (i) balanced structural aberrations such as translocations between sex chromosomes and autosomes; (ii) unbalanced structural aberrations such as deletions or isodicentrics, both [idic(Yp)] and [idic(Yq)]. We studied 13 subjects bearing Y chromosome aberrations. Each patient underwent seminal fluid examination, andrological inspection, hormone study, testicular ultrasound, conventional and molecular cytogenetic analysis and study of Y chromosome microdeletions. Comparison of genotype and sperm phenotype in infertile patients with various Y chromosome aberrations revealed the key role of meiotic pairing defects in arresting spermatogenesis, both in the presence and in the absence of azoospermic factor microdeletions and cell mosaicism. The failure of meiosis and, in consequence, spermatogenesis may be a result of the failure to inactivate the X chromosome in the meiotic prophase, which is necessary for normal male spermatogenesis to take place.

Chromosome instability in T-cells cultured in the presence of pancuronium or fentanyl.

BACKGROUND: Genomic instability is recognized as a cause of cellular apoptosis and certain drugs that exhibit a proapoptotic effect are also able to induce chromosome damage. Since we found in recent experiments that drugs such as pancuronium and fentanyl exerted an apoptogenic effect on T cells, we studied the capacity of those agents to promote chromosome instability, i.e. chromosome aberrations (CA) and telomeric associations (tas) in peripheral blood lymphocytes. METHODS: Lymphocytes from healthy donors were cultured with pancuronium or fentanyl, using two different concentrations for each drug: 20 and 200 ng/ml for pancuronium and 10 and 30 ng/ml for fentanyl, respectively. Cells were exposed to each concentration of these drugs either for 24 or 48 h. The higher concentration chosen was the same at which we detected the proapoptotic effect in our previous works. Cytogenetic analysis was performed by means of a standard technique and chromosome aberrations or telomeric associations were blindly evaluated by two independent observers. RESULTS: The chromosome aberrations we observed in treated cells were not significantly different from control lymphocytes. However, an unusual rate of telomeric associations (P < 0.001) was detected in cells exposed to both pancuronium and fentanyl, at each concentration tested and at each exposure time of the study. CONCLUSIONS: Fentanyl and pancuronium do not have a direct clastogenic effect on T cultures, but at the same concentrations at which we demonstrated their apoptogenic power, these drugs are able to increase genomic instability through inducing an elevated rate of telomeric associations. Such a capacity could exploit in peripheral T cells the same mitochondrion-mediated signal pathway of apoptosis death.

Telomerase activity, apoptosis and cell cycle progression in ataxia telangiectasia lymphocytes expressing TCL1.

Individuals affected by ataxia telangiectasia (AT) have a marked susceptibility to cancer. Ataxia telangiectasia cells, in addition to defects in cell cycle checkpoints, show dysfunction of apoptosis and of telomeres, which are both thought to have a role in the progression of malignancy. In 1-5% of patients with AT, clonal expansion of T lymphocytes carrying t(14;14) chromosomal translocation, deregulating TCL1 gene(s), has been described. While it is known that these cells can progress with time to a frank leukaemia, the molecular pathway leading to tumorigenesis has not yet been fully investigated. In this study, we compared AT clonal cells, representing 88% of the entire T lymphocytes (AT94-1) and expressing TCL1 oncogene (ATM(-) TCL1(+)), cell cycle progression to T lymphocytes of AT patients without TCL1 expression (ATM(-) TCL1(-)) by analysing their spontaneous apoptosis rate, spontaneous telomerase activity and telomere instability. We show that in ATM(-) TCL1(+) lymphocytes, apoptosis rate and cell cycle progression are restored back to a rate comparable with that observed in normal lymphocytes while telomere dysfunction is maintained.

Telomeric associations and chromosome instability in ataxia telangiectasia T cells characterized by TCL1 expression.

T-cell tumors in ataxia telangiectasia (AT), such as T-PLL/T-CLL, are first preceded by the development of a large clone of T-lymphocytes, characterized by chromosomal rearrangements, which usually involve specific regions such as the 14q11 region. Malignancy develops years later, after additional chromosomal changes resulting from the genomic instability consequent to ATM disruption and to the activation of the TCL1 oncogene. Here we report the results of a cytogenetic follow-up of an AT patient (AT94-1), still without signs of hematological abnormalities, bearing a T-lymphocyte clone characterized by the t(14;14)(q11;q32) rearrangement and having TCL1 expression. We demonstrated that in clonal cells TCL1 expression correlates with increasing genomic instability and in time this mainly induces chromosomal rearrangements and telomeric associations (tas). Chromosome 21 is not randomly involved; in particular, an i(21q) indicates that it is a subclone prone to additional genetic changes and could represent an early chromosomal rearrangement involved in tumorigenesis. With regard to the increase in tas, we observed that: (i) it is inversely correlated with the proliferative ability of AT94-1 lymphocytes in PHA-stimulated short-term cultures (cell aging in vitro); (ii) this increase is not due to changes either in cell radiosensitivity (measured as bleomycin (BML)-sensitivity) or due to an illegitimate recombination (measured as adriamycin-sensitivity), which may not be sufficient for tumor development.

Chromosomal alterations and male infertility.

Reduced male fertility can be caused by genetic factors affecting gamete formation or function; in particular, chromosome abnormalities are a possible cause of male subfertility as shown by their higher frequency in infertile men than in the general male population. Meiotic studies in a number of these males have shown spermatogenesis breakdown, often related to alterations in the process of chromosome synapsis. Indeed, any condition that can interfere with X-Y bivalent formation and X-chromosome inactivation is critical to the meiotic process; furthermore, asynapsed regions may themselves represent a signal for the meiotic checkpoint that eliminates spermatocytes with synaptic errors. We performed cytogenetic, hormonal and seminal studies in 333 infertile patients selected because azoospermic, severely oligozoospermic or normozoospermic with failure to fertilize the partner's oocytes in an in vitro fertilization (IVF) program. Our findings: 1) confirm the high incidence of chromosomal anomalies among infertile males; 2) highlight the relevance in male infertility of quantitative/positional modifications of the constitutive heterochromatin; and 3) underline the relevance of cooperation between andrologists and cytogenetists prior to every kind of assisted reproduction, above all prior to intracytoplasmic sperm injection, in which selective hurdles eliminating abnormal germ cells are bypassed.

Effects of poly(ADP-ribose) polymerase inhibition on cell death and chromosome damage induced by VP16 and bleomycin.

Poly(ADP-ribose) polymerase (PARP) is a DNA-binding protein involved in cellular response to various genotoxic agents. To understand the role of PARP in the mechanisms which lead from specific DNA damage to cell death, we studied the effects of PARP inhibition in human lymphoblasts damaged with bleomycin (BLM) and VP16. These agents can induce DNA breakage but through different mechanisms, enabling the study of the different effects of PARP in inducing apoptosis in damaged cells. We demonstrate that in lymphoblasts VP16 treatment induces apoptosis to a greater extent than BLM treatment, and that PARP inhibition reduces VP16-induced apoptosis whereas it has no effect on BLM-induced apoptosis. After VP16 treatment with PARP inhibition, a reduction in the depletion of the proliferative compartment and a G2/M phase arrest are observed. Therefore, the increase in cell viability and the reduction in chromosome damage may both be the result of a prolonged DNA repair time. Hence, PARP appears to play a significant role in VP16-induced apoptosis and not in BLM-induced apoptosis. Since apoptosis is important in tumor treatment these findings might be useful when considering the combined employment of PARP inhibition with antineoplastic drugs.

Effects of topoisomerase II inhibition in lymphoblasts from patients with progeroid and "chromosome instability" syndromes.

DNA topoisomerase II is involved in DNA topologic changes through the formation of a cleavable complex. This is stabilized by the antitumor drug VP16, which results in DNA breakage, aberrant recombination, and cell death. In this work, we compare the chromosomal damage induced by VP16 with that induced by bleomycin (BLM) in lymphoblasts from patients affected by the chromosome breakage syndromes ataxia telangiectasia (AT), xeroderma pigmentosum (XP), and Bloom syndrome (BS), and by the progeroid syndromes Werner (WS) and Cockayne (CS). Patients affected by AT, XP, BS, and WS have a greatly enhanced risk of developing cancer. The results show that AF and WS cells are hypersensitive to VP16, as revealed in the higher proportion of metaphases showing exchange figures and more than two breaks. All lines except AT and one CS line showed normal sensitivity to BLM. Our data on the sensitivity to VP16 of all these mutant cells underline the fact that VP16 damage is amplified only in cells that have abnormal illegitimate recombination (i.e., AT and WS).

VP16 hypersensitivity and increased faulty recombination in ataxia telangiectasia lymphocytes characterized by the tandem translocation t(14;14)(q11;q32).

Ataxia telangiectasia (AT) patients show variable degrees of immunodeficiency and a higher than normal predisposition to lymphoid malignancies. AT cells are characterized by spontaneous chromosome instability resulting in chromosome breakage and in non random chromosome rearrangements. Sequential cytogenetic studies on T-lymphocytes from an AT patient showed the progressive development of a clone bearing a tandem translocation t(14;14)(q11;q32). The abnormal clone had spontaneous chromosome rearrangements. Compared to non clonal cells, the abnormal clone displayed a higher frequency of spontaneous chromosome rearrangements. In only the clonal cells we observed two particular and predominant rearrangements: isodicentric chromosomes and telomeric associations which may derive from faulty recombination. Chromosome instability induced by the etoposide VP16, a DNA topoisomerase II inhibitor, was evaluated in terms of chromosome breakage and SCE frequency. T-lymphocytes from the AT patient showed hypersensitivity to VP16 significantly higher than normal T-lymphocytes. The chromosome instability induced by VP16 is significantly higher in clonal than in non clonal cells, whilst the chromosome instability induced by the radiomimetic drug bleomycin is not significantly different in the two AT lymphocyte subpopulations. The different spontaneous chromosome instability in clonal and non clonal cells together with their different behavior after treatment with only VP16, suggest that clonal cells bearing the tandem translocation could have increased faulty recombination. Given the presence of translocations t(14;14)(q11;q32) in T-prolymphocytic leukemias and T-cell tumors of non AT patients, our findings suggest that VP16 could be considered an antineoplastic treatment particularly indicated in these patients.

TCL1 oncogene activation in preleukemic T cells from a case of ataxia-telangiectasia.

The TCL1 oncogene on human chromosome 14q32.1 is involved in chromosome translocations [t(14;14)(q11;q32.1) and t(7;14)(q35;q32.1)] and inversions [inv14(q11;q32.1)] with TCR alpha/beta loci in T-cell leukemias, such as T-prolymphocytic (T-PLL). It is also involved in T-acute and -chronic leukemias arising in cases of ataxia-telangiectasia (AT), an immunodeficiency syndrome. Similar chromosomal rearrangements occur also in the clonally expanded T cells in AT patients before the appearance of the overt leukemia. We have analyzed the expression of TCL1 mRNA and protein in peripheral blood lymphocytes (PBLs) from four AT cases and from healthy controls. We found that the TCL1 gene was overexpressed in the PBLs of an AT patient with a large clonal T-cell population exhibiting the t(14;14) translocation but not in the lymphocytes of the other cases. Fluorescence in situ hybridization of the TCL1 genomic locus to lymphocyte metaphases from the AT patient with the T-cell clonal expansion showed that the breakpoint of the t(14;14) translocation lies within the TCL1 locus and is accompanied by an inverted duplication of the distal part of chromosome 14. These data indicate that TCL1 is activated in preleukemic clonal cells as a consequence of chromosome translocation involving sequences from the TCR locus at 14q11. Deregulation of TCL1 is the first event in the initiation of malignancy in these types of leukemias and represents a potential tool for clinical evaluation.

Expression of genes carried by pR plasmid in damaged E. coli and mouse cells.

In LTA mouse cells pR plasmid constitutively expresses itself resulting in protection against typical SOS inducers (UV, 4NQO) and in sensitization to different DNA-damaging agents (MNNG, cisDDP, BLM and geneticin (G418). The pR sensitizing effect is specific to mammalian cells, since the plasmid can only protect prokaryotic cells against the damaging agents tested. The pR protecting effect requires the expression of both the uvp1 and uvp2 (mucAB) regions in bacteria as well as in mouse cells. The coordinated function of these regions could result in protection against typical SOS inducers through an SOS/SOS-like pathway. The sensitization conferred by pR plasmid depends mostly on the expression of the mucAB genes, as shown by the survival of mouse cells transfected with different pR::Tn5 mutants. In particular, BLM and G418 survival data demonstrate that, inserted into the pR plasmid, the ble and neo genes of the Tn5 transposon express themselves. This was confirmed by the presence of Tn5 transcripts in untreated mouse cells. The comparison between the pR effects in bacterial and mouse cells shows that during evolution the repair pathways against UV damage are better conserved than those against other kinds of damage.

Neurological and cytogenetic study in early-onset ataxia-telangiectasia patients.

The clinical diagnosis of ataxia-telangiectasia (AT) is difficult before the age of 4 years. We report clinical and cytogenetic data on three early-onset, early-diagnosed AT patients at the age of 12, 18 and 22 months, respectively. Postural instability of the trunk, characterized by motor impersistence, was the earliest neurological sign detected as early as 1 year of life. Dystonic movements and postures of arms and trunk and a subtle disorder of eye movement (blinking before gaze changing, increased latency and dysmetry of saccades) were observed during the 2nd year of life. All patients exhibited an unusual temper tantrum. We also observed an increased bleomycin-induced chromosomal instability in patient's cells in the early stages of the disease before all the clinical hallmarks were apparent. Our data suggest that detection of clinical indications, leading to early laboratory confirmation of AT, can reduce the age at diagnosis.

Heterogeneity in ataxia-telangiectasia: classical phenotype associated with intermediate cellular radiosensitivity.

We identified a subgroup of ataxia-telangiectasia (AT) patients (2 sibs and 1 unrelated case) characterized by typical clinical manifestations of the disease and cellular radiosensitivity intermediate between classical AT and normal subjects. Our data and a literature review of the intermediate radiosensitivity AT cases show that radioresistant DNA synthesis, cellular radiosensitivity (measured in terms of survival and chromosome breakage), and the clinical hallmarks behave independently. This raises a number of interesting questions about the correlation between radiobiological and clinical features, and about the nature of the AT gene(s).

Human cells (normal and ataxia telangiectasia) transfected with pR plasmid are hypersensitive to DNA strand-breaking agents.

Ataxia telangiectasia (AT) cells are known to be hypersensitive to ionizing radiations and to drugs such as bleomycin and epipodophyllotoxin VP16, a topoisomerase II poison. Both of these produce DNA double-strand breaks even if through different mechanisms. In this work we analyzed the sensitivity to bleomycin and to epipodophyllotoxin of AT cells after transfection with pR plasmid. This plasmid, interacting with bacterial SOS repair pathways, expresses itself in mammalian cells conferring cell resistance to the SOS inducers UV and 4NQO and cell sensitivity to different drugs such as bleomycin. This effect is presumably due to the interaction of pR products with double-strand breaks. Our findings indicate that pR plasmid, in both AT lines tested (AT5BIVA fibroblasts and ATL6 lymphoblasts), expresses itself (increasing UV protection) and amplifies the already enhanced AT cell sensitivity to both bleomycin and VP16.

The rep region of pR plasmid regulates the expression of SOS system.

By using an artificial hybrid between phage lambda and the pR plasmid, we have shown that the rep region of the pR plasmid encodes a function which regulates the expression of the muc genes (plasmid genes that are under the negative control of lexA and responsible for an increased rate of spontaneous mutagenesis and resistance to UV and chemicals). Expression of the muc genes were monitored by a fusion between the muc promoter and the lacZ structural gene. When E. coli cells containing such a fusion are infected by the hybrid lambda pR phasmid, beta-galactosidase activity is enhanced, indicating that pR encodes an antagonist of lexA. By deletion mapping we have located the gene encoding the antagonist of lexA (bat) in the rep region of the plasmid. The bat gene product can also antagonize the lambda cI repressor as shown by the observation that lambda pR phasmids are virulent on a homoimmune lysogen. We have exploited this latter property to carry out genetic and functional analysis of the bat region. This region is organized as a classical operon where the expression of the bat structural gene is negatively regulated by a repressor gene that encodes a proteic product.

The pR plasmid: a tool for discriminating between DNA lesions induced by different types of cytotoxic agents in cultured mammalian cells.

The LA-D cells, obtained by cotransformation of LTA mouse cells (tk- aprt-) with pR plasmid and with tk gene as selective marker, are significantly more resistant to UV light and 4-nitroquinoline-N-1-oxide than LTA control cells. In this work, we report that the LA-D cells exhibit different degrees of response to various DNA-damaging agents: wild-type survival to mitomycin, increased sensitivity to bleomycin, cis-diamminedichloroplatinum and N-methyl-N'-nitro-N-nitrosoguanidine. The pR plasmid could, therefore, play an important role in the DNA-repair mechanisms that modulate the cytotoxic effect of the DNA-inhibitory agents. The possible interactions between pR plasmid products and the different repair enzymes involved are discussed.