A global non-invasive methodology for the phenotyping of potato under water deficit conditions using imaging, physiological and molecular tools.

BACKGROUND: Drought is a major consequence of global heating that has negative impacts on agriculture. Potato is a drought-sensitive crop; tuber growth and dry matter content may both be impacted. Moreover, water deficit can induce physiological disorders such as glassy tubers and internal rust spots. The response of potato plants to drought is complex and can be affected by cultivar type, climatic and soil conditions, and the point at which water stress occurs during growth. The characterization of adaptive responses in plants presents a major phenotyping challenge. There is therefore a demand for the development of non-invasive analytical techniques to improve phenotyping. RESULTS: This project aimed to take advantage of innovative approaches in MRI, phenotyping and molecular biology to evaluate the effects of water stress on potato plants during growth. Plants were cultivated in pots under different water conditions. A control group of plants were cultivated under optimal water uptake conditions. Other groups were cultivated under mild and severe water deficiency conditions (40 and 20% of field capacity, respectively) applied at different tuber growth phases (initiation, filling). Water stress was evaluated by monitoring soil water potential. Two fully-equipped imaging cabinets were set up to characterize plant morphology using high definition color cameras (top and side views) and to measure plant stress using RGB cameras. The response of potato plants to water stress depended on the intensity and duration of the stress. Three-dimensional morphological images of the underground organs of potato plants in pots were recorded using a 1.5 T MRI scanner. A significant difference in growth kinetics was observed at the early growth stages between the control and stressed plants. Quantitative PCR analysis was carried out at molecular level on the expression patterns of selected drought-responsive genes. Variations in stress levels were seen to modulate ABA and drought-responsive ABA-dependent and ABA-independent genes. CONCLUSIONS: This methodology, when applied to the phenotyping of potato under water deficit conditions, provides a quantitative analysis of leaves and tubers properties at microstructural and molecular levels. The approaches thus developed could therefore be effective in the multi-scale characterization of plant response to water stress, from organ development to gene expression.

Estimation of time since death by vitreous humor hypoxanthine, potassium, and ambient temperature.

Measurement of vitreous humor potassium (K(+)) has since the 1960s been recognized as an adjunct for estimation of time since death. In 1991 we introduced hypoxanthine (Hx) as a new marker. Furthermore we demonstrated that time since death estimation was more accurate when ambient temperature was included in the calculations, both for K(+) and for Hx. In this paper we present a refined method. The subjects consist of 132 cases with known time of death and ambient temperature. One sample from each subject was used in the calculations. Vitreous humor Hx levels were available in all subjects, while K(+) was measured in 106 of the subjects, due to insufficient volume of vitreous humor. Linear regression analysis was applied to model the correlation between vitreous humor Hx and K(+), taking the interactions with temperature into consideration. The diagrams published in 1991, which also included ambient temperature, estimated median time since death with range between the 10th and 90th percentile, whereas the linear regression analysis presented in this paper estimates mean time since death with a corresponding 95% interval of confidence. We conclude that time since death may be estimated with relatively high precision applying vitreous humor Hx and K(+) concentrations combined with ambient temperature.

Is a CIS phenotype apparent in children with Disorders of Sex Development? Milder testicular dysgenesis is associated with a higher risk of malignancy.

All malignant testicular germ cell tumors (TGCT) of adult men are preceded by an in situ stage (CIS) of protracted evolution. The adult CIS is well characterized, but there is debate on the phenotype of infantile CIS, its distinction from delayed maturation of germ cells and prognostic potential. A large series of 43 patients with Disorders of Sex Development (DSD) and dysgenetic testes (90% ranging from neonates to 12 years, mean age 4.7 years), was studied by quantifying dysgenetic features, degree of germ cell abnormalities/atypia (GCA), expression of OCT 3/4 (a pluripotency-undifferentiation marker), germ cell ploidy and evolution to CIS and invasive TGCT. Findings were compared with those of normal testes. The type of gonads present defined three groups of patients: bilateral testes (BT-DSD, n = 21), one testis and one streak gonad (CT-DSD, C for combined, n = 13), and ovarian-testicular combinations (OT-DSD, n = 9). There were 5 boys with infantile CIS, bilateral in 3 (total of 8 infantile CIS) and two patients with adult CIS, bilateral in one (total of 3 adult CIS). Two patients had bilateral seminomas one at 12-17 and the other at 23 years. Histological dysgenesis was significantly higher in CT-DSD (p < 0.05), that had only 1 CIS. The highest frequency of GCA was in BT-DSD (p < 0.05), which coincided with a total of 11CIS + Seminomas. In all patients, aneuploidy was significantly higher (63%) than diploidy (p < 0.02), and GCA were more frequent in aneuploid than in diploid samples (p < 0.02). All CIS and TGCT were OCT 3/4 positive. Finally, there was a significant association between the triad Aneuploidy + GCA + OCT 3/4 positivity and the incidence of CIS (Fisher Exact test p < 0.002, relative risk 7.0). The degree of testicular dysgenesis (derived from abnormal organization of Sertoli cells in fetal testicular cords) is inversely related to the incidence of CIS. Our data demonstrate that the combined use of OCT 3/4 expression, quantification of germ cell abnormalities-atypia and ploidy in dysgenetic testes can satisfactorily identify infantile CIS with high risk of malignant evolution and set it aside from delayed germ cell maturation with lower or nil neoplastic potential.

Are substitutions in the first hypervariable region of the mitochondrial DNA displacement-loop in sudden infant death syndrome due to maternal inheritance?

AIM: To investigate whether all substitutions in the first hypervariable region (HVR1) in sudden infant death syndrome (SIDS) can be recovered along the maternal line of the family (inherited), or whether SIDS victims have new substitutions compared to maternal relatives (somatic mutations) that may be related to environmental factors. METHODS: Seventy-one SIDS/mother pairs, including 11 families with SIDS, mother and mother's relatives and/or SIDS siblings, were studied. The HVR1 sequence was recorded in the base-pair range 16056-16400. The recorded HVR1 sequence was compared with the Cambridge sequence, and differences were recorded as substitutions. The substitution pattern in the SIDS victims was compared with the pattern found in family members along the maternal line. RESULTS: All the substitutions found in SIDS victims could be traced in the maternal line of the family; in 5 cases this was observed through three generations, and in 3 cases through four generations. DISCUSSION: In patients with known mitochondrial (mt) DNA disease, a large number of sequence variants have been found in the D-loop region. Substitutions in the D-loop may be part of a haplotype with mutations elsewhere in the mtDNA. CONCLUSION: HVR1 substitutions in SIDS victims are hereditary and not due to somatic mutations.

Molecular genetic analysis of two human sperm fibrous sheath proteins, AKAP4 and AKAP3, in men with dysplasia of the fibrous sheath.

Dysplasia of the fibrous sheath (DFS) is characterized by male infertility, asthenozoospermia, and morphologically abnormal flagella that possess a severely malformed fibrous sheath. In many cases, DFS is familial, suggesting a genetic component. Human AKAP4 and AKAP3 are structural proteins of the fibrous sheath that also function to anchor protein kinase A to this structure via the regulatory subunit of the kinase. We hypothesized that defects in either AKAP4 or AKAP3 might cause DFS. No quantitative or qualitative differences between patients with DFS and normal controls were detected when sperm proteins were analyzed by either silver staining or immunoblot analysis using antibodies raised against AKAP4 and AKAP3. Additionally, AKAP4 and AKAP3 from DFS sperm retained the ability to bind the regulatory subunit of protein kinase A. Localization at the light and electron microscopic levels showed that AKAP3 and AKAP4 localized correctly to the FS of the amorphous flagellum in DFS sperm. Partial sequence analysis of the AKAP4 and AKAP3 genes in patients with DFS did not identify any significant alterations in potential AKAP4/AKAP3 binding regions, suggesting that the two proteins interact normally in DFS sperm. Our results did not find evidence to support the hypothesis that mutations in either gene are responsible for DFS in humans.

Cytology of transitional-cell carcinoma of the urinary bladder: diagnostic yield and histologic basis.

The diagnostic yield of cytology in histologically proven transitional-cell carcinoma (TCC) of the urinary bladder has been studied in 100 cases. Cytohistologic correlation rates were 20 percent, 61.7 percent, and 92.8 percent, respectively, for grade 1, 2, and 3 tumors. When further evaluated, G2 cases were segregated into 2 subcategories, G2a and G2b, based on histologic preservation of nuclear polarity, pleomorphism, and other cellular irregularities. Correlation rates were rather low for G2a cases (6/18, 33%) and high for G2b cases, (23/29; 79%). The prevalence of atypical cells was 2 (11.1%) cases in G2a and 16 (55.2%) cases in G2b. The results of this study confirm that cytology has an extremely varying diagnostic yield in urinary bladder TCC. Greater cell exfoliation, increased atypia, and a tendency to infiltration of G2b and G3 cases probably account for the higher diagnostic yield detected in these groups.

Primary liver cancer in non-cirrhotic liver. Epidemiological study based on autopsies performed in Trieste, Italy and Kurume, Japan.

This study, aimed at elucidating the epidemiological features of primary liver carcinoma developing in non-cirrhotic livers, was based on 25,103 autopsies performed between 1975 and 1984 in Trieste, Italy. These autopsies correspond to approximately 70% of all deaths that occurred in this area. Various factors allegedly related to carcinomas were analysed in reference to our previous study on cirrhotic livers and in comparison with 5,603 autopsies in Kurume, Japan. There were 28 cases of hepatocellular carcinoma (HCC), 16 of cholangiocellular carcinoma (CCC) not associated with cirrhosis in Trieste, and 48 HCC and 19 CCC in Kurume. On the basis of our findings, it was concluded that cirrhosis, regardless of its cause, is the main pathogenetic factor in HCC; it is responsible for a much higher frequency (14.2:1) than in non-cirrhotic livers, as well as for early occurrence of tumours (an average of 6 years earlier in cirrhotic liver) in Trieste. Patients in Trieste were older than those in Japan, and the frequency of HCC among all autopsies was much greater in the latter. By contrast, the influence of cirrhosis on cholangiocellular carcinoma (CCC) was negligible, as such association appeared purely coincidental or absent. The incidence of CCC among autopsies was greater in Japan. Our data on CCC were not sufficient to demonstrate any clear aetiopathogenetic association between this tumour and alcohol abuse and hepatitis B virus (HBV) infection, except for a possible aetiological role of gallstones. The frequency of CCC relative to HCC was greater in Trieste than in Japan; the incidence of HCC was much less in Trieste, whereas CCC was more frequent in Japan.

Occurrence of liver cirrhosis among autopsies in Trieste.

The reports of 26,879 autopsies performed at the Institute of Pathological Anatomy at the University of Trieste during 1876-85 (70% of all deaths that occurred in the Province) were examined, and 2563 cases of liver cirrhosis were found. Analysis of the sample allowed us to make the following conclusions: (i) The prevalence of cirrhosis at autopsy is high in Trieste and shows no tendency to decrease, as has been inferred by some clinical studies. (ii) The increasing average age at death over the decade studied appears to be unrelated to the new, early treatments adopted for hepatopathic patients, since a similar yearly increase in mean age at death was seen for the whole population of the Province. The combination of a high incidence of cirrhosis and increasing average age of patients will probably result in an increasing occurrence of hepatocellular carcinoma. (iii) The observed male:female ratio (2.3) is analogous to that of alcohol drinkers in the Province and thus suggests a role of alcohol abuse in the development of cirrhosis. The distribution of markers of hepatitis B virus in the population of Trieste, which is very similar in the two sexes, supports this hypothesis.

[Endometrial hyperplasia in the province of Trieste]. / Le iperplasie dell'endometrio nella provincia di Trieste.

1103 cases of endometrial hyperplasia were diagnosed between January 1, 1978 and December 31, 1987 at the Department of Anatomy and Pathological Histology of the University of Trieste. Of these, 58% were cases of cystic glandular hyperplasia, 38% of adenomatous hyperplasia and 4% of atypical hyperplasia. Subdividing the period examined into two five-year periods, it was found that the rate of incidence of endometrial hyperplasias was more than doubled, this increase having been caused above all by cases of cystic hyperplasia. Distribution by age classes of subjects suffering from different hyperplastic forms suggests that cystic glandular hyperplasia is not a precursor of the adenomatous form, whereas the latter and atypical hyperplasia might constitute a continuous spectrum of potentially precancerous lesions.