Influence of exposure to pesticides on telomere length and pregnancy outcome: Diethylphosphates but not Dimethylphosphates are associated with accelerated telomere attrition in a Palestinian cohort.

Exposure to environmental pesticides during pregnancy is associated with adverse health outcomes such as low birth weight and impaired neuro-development. In this study, we assessed maternal leukocyte telomere lengths (TL) in Palestinian pregnant women and compared the data with urinary organophosphate concentrations, demographic, lifestyle and dietary factors, birth weight, body length, gestational age, and head circumference. Women with high urine levels of creatinine adjusted diethylphosphate(DE)derived pesticide metabolites DEP, DETP or DEDTP had shorter telomeres (p = 0.05). Women living in proximity to agricultural fields had shorter telomeres compared to women not living in proximity to agricultural fields (p = 0.011). Regular consumption of organic food was associated with shorter telomeres (p = 0.01), whereas the consumption of other vegetables such as artichokes was rather associated with longer telomeres. By contrast, urine levels of dimethylphosphate(DM)-derived pesticide metabolites DMTP and DMDTP were associated with lower birth weight (p = 0.05) but not with shrter telomeres. In conclusion organophosphate pesticides and living in proximity to agriculture are associated with shorter TL, likely due to higher consumption of contaminated fruits and vegetables and/or the transport of pesticides to non-treatment sites. DE and DM substituted pesticides seem to have different effects on telomeres and development.

CD11c-positive cells from brain, spleen, lung, and liver exhibit site-specific immune phenotypes and plastically adapt to new environments.

The brain's immune privilege has been also attributed to the lack of dendritic cells (DC) within its parenchyma and the adjacent meninges, an assumption, which implies maintenance of antigens rather than their presentation in lymphoid organs. Using mice transcribing the green fluorescent protein under the promoter of the DC marker CD11c (itgax), we identified a juxtavascular population of cells expressing this DC marker and demonstrated their origin from bone marrow and local microglia. We now phenotypically compared this population with CD11c/CD45 double-positive cells from lung, liver, and spleen in healthy mice using seven-color flow cytometry. We identified unique, site-specific expression patterns of F4/80, CD80, CD86, CX3CR1, CCR2, FLT3, CD103, and MHC-II. Furthermore, we observed the two known CD45-positive populations (CD45(high) and CD45(int) ) in the brain, whereas liver, lung, and spleen exhibited a homogeneous CD45(high) population. CD11c-positive microglia lacked MHC-II expression and CD45(high) /CD11c-positive cells from the brain have a lower percentage of MHC-II-positive cells. To test whether phenotypical differences are fixed by origin or specifically develop due to environmental factors, we transplanted brain and spleen mononuclear cells on organotypic slice cultures from brain (OHSC) and spleen (OSSC). We demonstrate that adaption and ramification of MHC-II-positive splenocytes is paralleled by down-regulation of MHC-II, whereas brain-derived mononuclear cells neither ramified nor up-regulated MHC-II in OSSCs. Thus, brain-derived mononuclear cells maintain their MHC-II-negative phenotype within the environment of an immune organ. Intraparenchymal CD11c-positive cells share immunophenotypical characteristics of DCs from other organs but remain unique for their low MHC-II expression.

Characterization of cholesterol homeostasis in telomerase-immortalized Tangier disease fibroblasts reveals marked phenotype variability.

We compared the consequences of an ABCA1 mutation that produced an apparent lack of atherosclerosis (Tangier family 1, N935S) with an ABCA1 mutation with functional ABCA1 knockout that was associated with severe atherosclerosis (Tangier family 2, Leu(548):Leu(575)-End), using primary and telomerase-immortalized fibroblasts. Telomerase-immortalized Tangier fibroblasts of family 1 (TT1) showed 30% residual cholesterol efflux capacity in response to apolipoprotein A-I, whereas telomerase-immortalized Tangier fibroblasts of family 2 (TT2) showed only 20%. However, there were a number of secondary differences that were often stronger and may help to explain the more rapid development of atherosclerosis in family 2. First, the total cellular cholesterol content increase was 2-3-fold and 3-5-fold in TT1 and TT2 cells, respectively. The corresponding increase in esterified cholesterol concentration was 10- and 40-fold, respectively. Second, 24-, 25-, and 27-hydroxycholesterol concentrations were moderately increased in TT1 cells, but were increased as much as 200-fold in TT2 cells. Third, cholesterol biosynthesis was moderately decreased in TT1 cells, but was markedly decreased in TT2 cells. Fourth, potentially atheroprotective LXR-dependent SREBP1c signaling was normal in TT1, but was rather suppressed in TT2 cells. Cultivated primary Tangier fibroblasts were characterized by premature aging in culture and were associated with less obvious biochemical differences. In summary, these results may help to understand the differential atherosclerotic susceptibility in Tangier disease and further demonstrate the usefulness of telomerase-immortalized cells in studying this cellular phenotype. The data support the contention that side chain-oxidized oxysterols are strong suppressors of cholesterol biosynthesis under specific pathological conditions in humans.

[Successful surgical excision of a melanoma and a rare peripheral nerve sheath tumor in 2 cattle]. / Erfolgreiche chirurgische Exzision eines Melanoms und eines seltenen peripheren Nervenscheidentumors bei 2 Rindern.

A heifer and a dairy cow were presented to our practice with cutaneous masses on the left side of their necks. Each mass had a diameter of approximately 20 cm. Both tumors had increased in size in recent weeks and were now prone to injuries from the stable equipment. Both animal owners agreed to surgical removal, which was performed under sedation and local anesthesia on a bovine treatment crush. The subsequent histopathological examinations of the extirpates revealed a melanocytoma in the young heifer and a cutaneous peripheral nerve sheath tumor (PNST) in the dairy cow. Both cases were benign tumors. The postoperative course was without complications and no recurrences were observed even more than a year later. No comparable tumors were found in related animals or in the offspring.

Laryngeal and oropharyngeal adenocarcinoma with pulmonary metastases in a common raven (Corvus corax).

A captive 15-year-old male common raven (Corvus corax) was presented for post-mortem examination. It had been previously presented to a local veterinarian due to a 3-4 weeks long history of abnormal respiratory sounds. Upon admission, the bird demonstrated severe dyspnea and a massive amount of mucous in the oropharynx. After symptomatic treatment, dyspnea deteriorated dramatically, and euthanasia was elicited because of poor prognosis. The necropsy revealed a 2.65 x 2.15 x 2.18 cm expansile and poorly delineated cauliflower-shaped mass around the glottis and extending inside the tracheal lumen. Additionally, a dilated salivary gland in the adjacent tissue and multifocal reddish-fleshy areas in the lung parenchyma were detected. Histopathological examination identified the mass as moderately differentiated, tubular adenocarcinoma with invasive growth and moderate to marked cellular atypia and numerous mitoses. The presumptive origin of the neoplasia was one of the salivary glands. Multiple metastases were identified in the lung both macroscopically and histologically. Bacterial culture and molecular testing for West Nile and Usutu viruses were negative. To the authors' knowledge, this is the first report of metastatic laryngeal and oropharyngeal adenocarcinoma in a common raven.

Use of Bacterial DNA Concentration in Ascites as a Marker for Spontaneous Bacterial Peritonitis.

Background and aims: Spontaneous bacterial peritonitis (SBP) is a common and serious complication in patients with decompensated cirrhosis. Precise quantification of bacterial DNA (bactDNA) and the related inflammatory response might add further information on the course of disease. The aim of the study was to evaluate the association between bactDNA, cytokine levels and clinical outcome. Methods: Ascites and serum samples of 98 patients with decompensated liver cirrhosis (42 with SBP and 56 without SBP) as well as serum samples of 21 healthy controls were collected. BactDNA in ascites and serum was detected and quantified by 16S rRNA PCR. Concentrations of IL-1ß, TNF-α, IL-6, IL-8 and IL-10 were measured by a LEGENDplexTM multi-analyte flow assay. Clinical data were collected and analyzed retrospectively. Results: BactDNA was detected more frequently in ascites of patients with SBP (n = 24/42; 57.1%) than in ascites of patients without SBP (n = 5/56; 8.9%; P < 0.001). Additionally, IL-6 levels in both ascites and serum were significantly higher in patients with SBP (ascites P < 0.001, serum P = 0.036). The quantity of bactDNA in ascites was strongly correlated with polymorphonuclear neutrophil count in ascites (r = 0.755; P < 0.001) as well as ascites IL-6 levels (r = 0.399; P < 0.001). Receiver operating characteristic (ROC) curve analysis to diagnose SBP provided an AUC of 0.764 (95% CI: 0.661-0.867) for serum IL-6 levels, an AUC of 0.810 (95% CI: 0.714-0.905) for ascites IL-6 levels, and an AUC of 0.755 (95% CI: 0.651-0.858) for bactDNA levels in ascites. Conclusions: The correlation between the amount of bactDNA and IL-6 confirms the pathophysiological relevance of bactDNA and IL-6 as potential biomarkers for the diagnosis of SBP.

Artificial Intelligence to Predict the BRAF V595E Mutation in Canine Urinary Bladder Urothelial Carcinomas.

In dogs, the BRAF mutation (V595E) is common in bladder and prostate cancer and represents a specific diagnostic marker. Recent advantages in artificial intelligence (AI) offer new opportunities in the field of tumour marker detection. While AI histology studies have been conducted in humans to detect BRAF mutation in cancer, comparable studies in animals are lacking. In this study, we used commercially available AI histology software to predict BRAF mutation in whole slide images (WSI) of bladder urothelial carcinomas (UC) stained with haematoxylin and eosin (HE), based on a training (n = 81) and a validation set (n = 96). Among 96 WSI, 57 showed identical PCR and AI-based BRAF predictions, resulting in a sensitivity of 58% and a specificity of 63%. The sensitivity increased substantially to 89% when excluding small or poor-quality tissue sections. Test reliability depended on tumour differentiation (p < 0.01), presence of inflammation (p < 0.01), slide quality (p < 0.02) and sample size (p < 0.02). Based on a small subset of cases with available adjacent non-neoplastic urothelium, AI was able to distinguish malignant from benign epithelium. This is the first study to demonstrate the use of AI histology to predict BRAF mutation status in canine UC. Despite certain limitations, the results highlight the potential of AI in predicting molecular alterations in routine tissue sections.

Establishment and Characterization of hTERT Immortalized Hutchinson-Gilford Progeria Fibroblast Cell Lines.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging syndrome caused by a dominant mutation in the LMNA gene. Previous research has shown that the ectopic expression of the catalytic subunit of telomerase (hTERT) can elongate the telomeres of the patients' fibroblasts. Here, we established five immortalized HGP fibroblast cell lines using retroviral infection with the catalytic subunit of hTERT. Immortalization enhanced the proliferative life span by at least 50 population doublings (PDs). The number of cells with typical senescence signs was reduced by 63 + 17%. Furthermore, the growth increase and phenotype improvement occurred with a lag phase of 50-100 days and was not dependent on the degree of telomere elongation. The initial telomeric stabilization after hTERT infection and relatively low amounts of hTERT mRNA were sufficient for the phenotype improvement but the retroviral infection procedure was associated with transient cell stress. Our data have implications for therapeutic strategies in HGP and other premature aging syndromes.

Histopathological Findings in the Cardiovascular System of Psittacidae in Routine Diagnostics.

Samples of 363 Psittacidae were included in this study with a focus on cardiovascular diseases. These were identified in 28.9% of the animals, with pericarditis and/or epicarditis and myocarditis representing approximately half of all lesions and bacteria being the most common infectious cause. Cardiac lymphoma was only seen in 5 birds, whereas degenerative vascular lesions were diagnosed in 26.7% of the cases. Histopathology in the context of clinical findings and complementary examination results is the most useful tool for the evaluation of cardiac diseases.

A conserved long-distance telomeric silencing mechanism suppresses mTOR signaling in aging human fibroblasts.

Telomeres are repetitive nucleotide sequences at the ends of each chromosome. It has been hypothesized that telomere attrition evolved as a tumor suppressor mechanism in large long-lived species. Long telomeres can silence genes millions of bases away through a looping mechanism called telomere position effect over long distances (TPE-OLD). The function of this silencing mechanism is unknown. We determined a set of 2322 genes with high positional conservation across replicatively aging species that includes known and candidate TPE-OLD genes that may mitigate potentially harmful effects of replicative aging. Notably, we identified PPP2R2C as a tumor suppressor gene, whose up-regulation by TPE-OLD in aged human fibroblasts leads to dephosphorylation of p70S6 kinase and mammalian target of rapamycin suppression. A mechanistic link between telomeres and a tumor suppressor mechanism supports the hypothesis that replicative aging fulfills a tumor suppressor function and motivates previously unknown antitumor and antiaging strategies.

Leukocyte Telomere Length in Patients with Multiple Sclerosis and Its Association with Clinical Phenotypes.

Aging is a significant factor influencing the course of multiple sclerosis (MS). Accelerated telomere attrition is an indicator of premature biological aging and a potential contributor to various chronic diseases, including neurological disorders. However, there is currently a lack of studies focusing on telomere lengths in patients with MS. We measured the average leukocyte telomere length (LTL) in biobanked DNA samples of 40 relapsing-remitting MS patients (RRMS), 20 primary progressive MS patients (PPMS), and 60 healthy controls using a multiplex quantitative polymerase chain reaction method. Changes in LTL over a period of >10 years were evaluated in a subset of 10 patients. Association analyses of baseline LTL with the long-term clinical profiles of the patients were performed using inferential statistical tests and regression models adjusted for age and sex. The cross-sectional analysis revealed that the RRMS group was characterized by a significantly shorter relative LTL, on average, as compared to the PPMS group and controls. Shorter telomeres at baseline were also associated with a higher conversion rate from RRMS to secondary progressive MS (SPMS) in the 10-year follow-up. The LTL decrease over time was similar in RRMS patients and PPMS patients in the longitudinal analysis. Our data suggest a possible contributory role of accelerated telomere shortening in the pathobiology of MS. The interplay between disease-related immune system alterations, immunosenescence, and telomere dynamics deserves further investigation. New insights into the mechanisms of disease might be obtained, e.g., by exploring the distribution of telomere lengths in specific blood cell populations.

[First description of canine leproid granuloma in Germany: 2 case reports]. / Erstbeschreibung des kaninen leproiden Granuloms in Deutschland: 2 Fallberichte.

Using cytology, histopathology, and DNA sequencing the diagnosis of canine leproid granuloma (CLG) was made in 2 dogs. The dogs were presented with nodular skin lesions on the head and pinnae. CLG is caused by nontuberculous mycobacteria that have not yet been finally classified. To date, this disease has been reported in Australia, New Zealand as well as North and South America, however no case reports have been published in Germany until now. In both cases, a combination of surgery and long-term drug administration (rifampicin, clarithromycin, doxycyclin and local application of clofazimin) was chosen and successfully eliminated the granulomas.

Telomere attrition and dysfunction: a potential trigger of the progeroid phenotype in nijmegen breakage syndrome.

BACKGROUND: Nibrin, as part of the NBN/MRE11/RAD50 complex, is mutated in Nijmegen breakage syndrome (NBS), which leads to impaired DNA damage response and lymphoid malignancy. RESULTS: Telomere length (TL) was markedly reduced in homozygous patients (and comparably so in all chromosomes) by ~40% (qPCR) and was slightly reduced in NBS heterozygotes older than 30 years (~25% in qPCR), in accordance with the respective cancer rates. Humanized cancer-free NBS mice had normal TL. Telomere elongation was inducible by telomerase and/or alternative telomere lengthening but was associated with abnormal expression of telomeric genes involved in aging and/or cell growth. Lymphoblastoid cells from NBS patients with long survival times (>12 years) displayed the shortest telomeres and low caspase 7 activity. CONCLUSIONS: NBS is a secondary telomeropathy. The two-edged sword of telomere attrition enhances the cancer-prone situation in NBS but can also lead to a relatively stable cellular phenotype in tumor survivors. Results suggest a modular model for progeroid syndromes with abnormal expression of telomeric genes as a molecular basis. METHODS: We studied TL and function in 38 homozygous individuals, 27 heterozygotes, one homozygous fetus, six NBS lymphoblastoid cell lines, and humanized NBS mice, all with the same founder NBN mutation: c.657_661del5.

Cytokeratin expression profiles of canine epithelial tissues.

Cytokeratins (CKs) are intermediate filaments of epithelial cells. In humans, different types of epithelia as well as their neoplasms show distinct CK expression profiles. The aim of this study was to establish a panel of CKs for the identification of specialized canine epithelia that can be integrated in a routine diagnostic setting. Immunohistochemistry was performed on 42 formalin-fixed paraffin-embedded (FFPE) canine unaltered tissues including all epithelial tissues by using an antibody panel detecting CKs 7, 8, 13, 14, 17, 19 and 20 and the pancytokeratin marker AE1/AE3. Using this antibody panel, a differentiation scheme for the identification of canine tissues was developed. This allowed the identification of 23 out of the 42 examined canine tissues and the distinction of 9 groups of specialized epithelia. The statistical validation revealed high variations in the immunoreactivity for CKs 7, 8, 14, 17 and 20 between the donor dogs. The antibody detecting CK 7 (OV-TL 12/13) showed a decrease in immunostaining after a fixation time of 3 and 4 days. To the best of the authors' knowledge this is the first study that characterizes all canine epithelial tissues for their expression of CKs 7, 8, 13, 14, 17, 19 and 20 and the pancytokeratin marker AE1/AE3. Results of this study are an important prerequisite for comparative histology and for the investigation into similarities/differences of the cytokeratin expression between normal and neoplastic epithelia. Since this study was performed on FFPE tissue, it can be included in the workflow of a routine diagnostic laboratory.

Chronic endometritis in an Asian elephant (Elephas maximus).

A 48-yr-old female Asian elephant with a history of pododermatitis developed recurrent hematuria beginning in 2002. Transrectal ultrasonography and endoscopic examination in 2004 identified the uterus as the source of hematuria and excluded hemorrhagic cystitis. Treatment with Desloreline implants, antibiotics, and homeopathic drugs led to an improved general condition of the elephant. In July 2005, the elephant was suddenly found dead. During necropsy, the severely enlarged uterus contained about 250 L of purulent fluid, and histopathology revealed ulcerative suppurative endometritis with high numbers of Streptococcus equi ssp. zooepidemicus and Escherichia coli identified on aerobic culture. Additional findings at necropsy included: multifocal severe pododermatitis, uterine leiomyoma, and numerous large calcified areas of abdominal fat necrosis. Microbiologic culture of the pododermatitis lesion revealed the presence of Streptococcus agalactiae, Streptococcus equi ssp. zooepidemicus, Staphylococcus sp., Corynebacterium sp., and Entercoccus sp.

Exposure of pregnant sows to deoxynivalenol during 35-70 days of gestation does not affect pathomorphological and immunohistochemical properties of fetal organs.

In order to evaluate the influence of deoxynivalenol (DON) on histomorphological and immunohistochemical parameters in the development of porcine fetuses, five pregnant sows were fed a control diet (0.15 mg DON/kg diet) and seven sows a contaminated diet (4.42 mg DON/kg diet) between days 35 and 70 of gestation. On day 70, fetuses were delivered by caesarean section and sows and fetuses were euthanized. Tissue samples of three fetuses from each sow were collected, fixed in formalin, and processed routinely for light microscopy and immunohistochemistry. At necropsy, no macroscopic lesions were observed in any organ of the fetuses. Histomorphological, immunohistochemical, and morphometrical parameters of the immune system, liver, and intestinal tract were examined. The following antibodies were used in the liver, spleen, lymph nodes, thymus, gut, and bone marrow to compare control- and DON-treated animals: (I) CD3 and CD79a (T and B lymphocytes differentiation); (II) myeloid/histiocyte antigen 387 (MAC) (identification of macrophages); (III) Ki-67 Antigen (Ki-67) (proliferation marker); (IV) p-p-38 mitogen-activated protein kinases (p-p38 MAPK) as well as caspase-3 (cas3) and caspase-9 (cas9) (enzymes of apoptosis cascade); (V) tumor necrosis factor-alpha (TNFα) (immune-related protein). The results of the study show that exposure of pregnant sows with DON between gestation days 35 and 70 causes no pathomorphologically or immunohistochemically detectable alterations in all fetal organs examined.

Therapeutic Targeting of Telomerase.

Telomere length and cell function can be preserved by the human reverse transcriptase telomerase (hTERT), which synthesizes the new telomeric DNA from a RNA template, but is normally restricted to cells needing a high proliferative capacity, such as stem cells. Consequently, telomerase-based therapies to elongate short telomeres are developed, some of which have successfully reached the stage I in clinical trials. Telomerase is also permissive for tumorigenesis and 90% of all malignant tumors use telomerase to obtain immortality. Thus, reversal of telomerase upregulation in tumor cells is a potential strategy to treat cancer. Natural and small-molecule telomerase inhibitors, immunotherapeutic approaches, oligonucleotide inhibitors, and telomerase-directed gene therapy are useful treatment strategies. Telomerase is more widely expressed than any other tumor marker. The low expression in normal tissues, together with the longer telomeres in normal stem cells versus cancer cells, provides some degree of specificity with low risk of toxicity. However, long term telomerase inhibition may elicit negative effects in highly-proliferative cells which need telomerase for survival, and it may interfere with telomere-independent physiological functions. Moreover, only a few hTERT molecules are required to overcome senescence in cancer cells, and telomerase inhibition requires proliferating cells over a sufficient number of population doublings to induce tumor suppressive senescence. These limitations may explain the moderate success rates in many clinical studies. Despite extensive studies, only one vaccine and one telomerase antagonist are routinely used in clinical work. For complete eradication of all subpopulations of cancer cells a simultaneous targeting of several mechanisms will likely be needed. Possible technical improvements have been proposed including the development of more specific inhibitors, methods to increase the efficacy of vaccination methods, and personalized approaches. Telomerase activation and cell rejuvenation is successfully used in regenerative medicine for tissue engineering and reconstructive surgery. However, there are also a number of pitfalls in the treatment with telomerase activating procedures for the whole organism and for longer periods of time. Extended cell lifespan may accumulate rare genetic and epigenetic aberrations that can contribute to malignant transformation. Therefore, novel vector systems have been developed for a 'mild' integration of telomerase into the host genome and loss of the vector in rapidly-proliferating cells. It is currently unclear if this technique can also be used in human beings to treat chronic diseases, such as atherosclerosis.

[Cutaneous infection with Orthopoxvirus bovis in a German Spaniel]. / Kutane Infektion mit Orthopoxvirus bovis bei einem Wachtelhund.

A 4-year-old female German Spaniel was presented with anorexia. Clinically, the dog showed papular to ulcerative lesions on the nasal planum and on the tongue. Hematological, bacteriological and mycological examinations did not contribute any evidence for the etiology of the lesions. Histopathological examination of skin biopsies revealed a proliferative dermatitis and folliculitis with hydropic degeneration of keratinocytes and cytoplasmatic inclusion bodies. Cowpox virus antigen was detected by immunohistochemistry, and electron microscopy showed pox virus particles in the cytoplasm of the epithelial cells. DNA of Orthopoxvirus bovis was identified by polymerase chain reaction. Consequently, in dogs with papular to ulcerative lesions in the face or on the tongue, infection with cowpoxvirus should be considered as an etiological differential diagnosis. Infected dogs represent a potential risk of infection for humans and other animals with close contact.

Telomere Length Is Not Related to Established Cardiovascular Risk Factors but Does Correlate with Red and White Blood Cell Counts in a German Blood Donor Population.

Telomere length (TL) is considered a marker of biological aging and has been associated with the presence of various coronary risk factors in patients. Much less is known about the relationships between TL and classic coronary risk factors in other populations. We measured TL in peripheral blood leukocytes of 343 middle-aged blood donors (mean age 40.2 ± 12.4 years; 201 men, 142 women) using quantitative polymerase chain reaction. Median TL was 0.86 (range: 0.48-1.85) relative TL units. In linear regression analyses with natural log-transformed T to S ratio as the dependent variable, there was a significant association with age (per year: beta = -0.007, p<0.001) and sex (males vs. females: beta = 0.075, p = 0.007) with longer telomeres in men. After adjusting for these two variables, we observed no association of TL with classic coronary risk factors including cholesterol (p = 0.36), triglyceride (p = 0.09), HDL-cholesterol (p = 0.26), LDL-cholesterol (p = 0.36), smoking (p = 0.97), and personal (p = 0.46) or family history (p = 0.63) of cardiovascular disease. However, we did find a significant positive association with white (p = 0.011) and red blood cell count (p = 0.031), hemoglobin (p = 0.014) and hematocrit (p = 0.013); we also found a borderline positive association with thrombocytes (p = 0.074). Positive associations remained significant for hemoglobin (p = 0.017), hematocrit (p = 0.023), and leukocytes (p = 0.009) in a subgroup with no reported vascular disease; associations were of borderline significance for erythrocytes (p = 0.053) and thrombocytes (p = 0.088) in this subgroup. The data do not support the concept that classic coronary risk factors contribute to telomere attrition in a blood donor population. However, telomere attrition may be a marker for reduced proliferation reserve in hematopoietic progenitor cells.

Cytometric patterns reveal growth states of Shewanella putrefaciens.

Bacterial growth is often difficult to estimate beyond classical cultivation approaches. Low cell numbers, particles or coloured and dense media may disturb reliable growth assessment. Further difficulties appear when cells are attached to surfaces and detachment is incomplete. Therefore, flow cytometry was tested and used for analysis of bacterial growth on the single-cell level. Shewanella putrefaciens was cultivated as a model organism in planktonic or biofilm culture. Materials of smooth and rough surfaces were used for biofilm cultivation. Both aerobic and anaerobic as well as feast and famine conditions were applied. Visualization of growth was also done using Environmental Scanning and Phase Contrast Microscopy. Bioinformatic tools were applied for data interpretation. Cytometric proliferation patterns based on distributions of DNA contents per cell corresponded distinctly to the various lifestyles, electron acceptors and substrates tested. Therefore, cell cycling profiles of S. putrefaciens were found to mirror growth conditions. The cytometric patterns were consistently detectable with exception of some biofilm types whose resolution remained challenging. Corresponding heat maps proved to be useful for clear visualization of growth behaviour under all tested conditions. Therefore, flow cytometry in combination with bioinformatic tools proved to be powerful means to determine various growth states of S. putrefaciens, even in constrained environments. The approach is universal and will also be applicable for other bacterial species.