A Reliable Method for Quantifying Plasma Cell-Free DNA Using an Internal Standard Strategy: Evaluation in a Cohort of Non-Pregnant and Pregnant Women.

The use of plasma cell-free DNA (cfDNA) as a useful biomarker in obstetric clinical practice has been delayed due to the lack of reliable quantification protocols. We developed a protocol to quantify plasma cfDNA using an internal standard strategy to overcome difficulties posed by low levels and high fragmentation of cfDNA. cfDNA was isolated from plasma samples of non-pregnant (NP, n = 26) and pregnant (P, n = 26) women using a commercial kit and several elution volumes were evaluated. qPCR parameters were optimized for cfDNA quantification, and several quantities of a recombinant standard were evaluated as internal standard. Absolute quantification was performed using a standard curve and the quality of the complete method was evaluated. cfDNA was eluted in a 50-µl volume, actin-ß (ACTB) was selected as the target gene, and qPCR parameters were optimized. The ACTB standard was constructed and 1000 copies were selected as internal standard. The standard curve showed R2 = 0.993 and E = 109.7%, and the linear dynamic range was defined between 102 and 106 ACTB copies/tube. Repeatability and reproducibility in terms of CV were 19% and up to 49.5% for ACTB copies per milliliter of plasma, respectively. The range of cfDNA levels was 428-18,851 copies/mL in NP women and 4031-2,019,363 copies/mL in P women, showing significant differences between the groups. We recommend the application of internal standard strategy for a reliable plasma cfDNA quantification. This methodology holds great potential for a future application in the obstetric field.

Punzi-loss:: a non-differentiable metric approximation for sensitivity optimisation in the search for new particles.

We present the novel implementation of a non-differentiable metric approximation and a corresponding loss-scheduling aimed at the search for new particles of unknown mass in high energy physics experiments. We call the loss-scheduling, based on the minimisation of a figure-of-merit related function typical of particle physics, a Punzi-loss function, and the neural network that utilises this loss function a Punzi-net. We show that the Punzi-net outperforms standard multivariate analysis techniques and generalises well to mass hypotheses for which it was not trained. This is achieved by training a single classifier that provides a coherent and optimal classification of all signal hypotheses over the whole search space. Our result constitutes a complementary approach to fully differentiable analyses in particle physics. We implemented this work using PyTorch and provide users full access to a public repository containing all the codes and a training example.

Glyphosate induces epithelial mesenchymal transition-related changes in human endometrial Ishikawa cells via estrogen receptor pathway.

Glyphosate based herbicides are the most commonly used herbicide in the world. We aimed to determine whether glyphosate (Gly) induces epithelial mesenchymal transition (EMT) - related changes in a human endometrial carcinoma cell line (Ishikawa cells), and whether the estrogen receptor (ER) pathway is involved in these changes. Ishikawa cells were exposed to Gly (0.2 µM and 2 µM) or 17ß-estradiol (E2: 10-9 M). We detected that Gly increased cell migration and invasion ability compared to vehicle, as did E2. Moreover, a down regulation of E-cadherin mRNA expression was determined in response to Gly, similar to E2-effects. These results show that Gly promotes EMT-related changes in Ishikawa cells. When an ER antagonist (Fulvestrant: 10-7 M) was co-administrated with Gly, all changes were reversed, suggesting that Gly might promote EMT-related changes via ER-dependent pathway. Our results are interesting evidences of Gly effects on endometrial cancer progression via the ER-dependent pathway.

Postnatal exposure to endosulfan affects uterine development and fertility.

Endosulfan is an organochlorine pesticide (OCP) used in large-scale agriculture for controlling a variety of insects and mites that attack food and non-food crops. Although endosulfan has been listed in the Stockholm Convention as a persistent organic pollutant to be worldwide banned, it is still in use in some countries. Like other OCPs, endosulfan is bioaccumulative, toxic and persistent in the environment. Human unintentional exposure may occur through air inhalation, dietary, skin contact, as well as, via transplacental route and breast feeding. Due to its lipophilic nature, endosulfan is rapidly absorbed into the gastrointestinal tract and bioaccumulates in the fatty tissues. Similar to other OCPs, endosulfan has been classified as an endocrine disrupting chemical (EDC). Endocrine action of endosulfan on development and reproductive function of males has been extensively discussed; however, endosulfan effects on the female reproductive tract have received less attention. This review provides an overview of: i) the fate and levels of endosulfan in the environment and human population, ii) the potential estrogenic properties of endosulfan in vitro and in vivo, iii) its effects on uterine development, and iv) the long-term effects on female fertility and uterine functional differentiation during early gestation.

Calidad de vida y dolor de cuello en estudiantes en ciencias de la salud / Qualidade de vida e dor de pescoço nosestudantes de ciências da saúde / Life quality and neck pain in health sciences students

Objetivos: Determinar fiabilidad y validez del instrumento,evaluar la CVRS, determinar la presencia e intensidad dedolor cervical, y analizar la relación entre dolor y calidad devida en la población de estudio. Material y Métodos: Estudioobservacional, de corte transversal en estudiantes de FCMUNC,con aplicación del cuestionario SF-36 y la Escala VisualAnalógica para intensidad de dolor. Las variables continuas ylas categóricas fueron analizadas con un nivel de significaciónde 0,05. Resultados: La población (n= 465) fue de 20,8 (±0,11) años de edad, en su mayoría mujeres (57,73%). El SF-36 mostró una fiabilidad y validez de constructo. La poblaciónpresentó mejores valores para el componente sumatoriofísico, siendo mayor en varones en las dimensiones: funciónfísica, dolor, vitalidad y salud mental (p < 0,05). El 86,06%de la población presentó dolor, siendo las mujeres las quelo percibieron mayor intensidad (p=0,0001) y correlacionócon la dimensión dolor del SF-36. Conclusión: El dolorcervical afecta la calidad de vida de los estudiantes, afectandoactividades que influyen en su rendimiento académico y suformación profesional.

Objectives: To determine the validity and reliability of the instrument, assess HRQL,determine the presence and intensity of cervical pain, and analyze the relationship betweenpain and quality of life in the studied population. Material and methods: Observational, crosssectional study in students from the School of Medicine, National University of Cordoba,using the SF-36 Survey and the Analogue Visual Scale for pain intensity. Continuous andcategorical variables were analyzed with significance level of 0.05. Results: The population(n= 465) was 20.8 (± 0.11) years old, mostly women (57.73%). The SF-36 showedconstruct reliability and validity. The population presented better values for the physicalsumming component; with the dimensions physical function, pain, vitality, and mentalhealth being greater in men (p < 0.05). The 86.06% of the population presented pain,women perceived it with greater intensity (p=0.0001) and correlated to pain dimensionfrom SF-36. Conclusion: Cervical pain affects students´ life quality, making an impact onthe activities related to their academic performance and professional training.

Growth, survival, and peptidolytic activity of Lactobacillus plantarum I91 in a hard-cheese model.

In this work, we studied the growth, survival, and peptidolytic activity of Lactobacillus plantarum I91 in a hard-cheese model consisting of a sterile extract of Reggianito cheese. To assess the influence of the primary starter and initial proteolysis level on these parameters, we prepared the extracts with cheeses that were produced using 2 different starter strains of Lactobacillus helveticus 138 or 209 (Lh138 or Lh209) at 3 ripening times: 3, 90, and 180 d. The experimental extracts were inoculated with Lb. plantarum I91; the control extracts were not inoculated and the blank extracts were heat-treated to inactivate enzymes and were not inoculated. All extracts were incubated at 34°C for 21 d, and then the pH, microbiological counts, and proteolysis profiles were determined. The basal proteolysis profiles in the extracts of young cheeses made with either strain tested were similar, but many differences between the proteolysis profiles of the extracts of the Lh138 and Lh209 cheeses were found when riper cheeses were used. The pH values in the blank and control extracts did not change, and no microbial growth was detected. In contrast, the pH value in experimental extracts decreased, and this decrease was more pronounced in extracts obtained from either of the young cheeses and from the Lh209 cheese at any stage of ripening. Lactobacillus plantarum I91 grew up to 8 log during the first days of incubation in all of the extracts, but then the number of viable cells decreased, the extent of which depended on the starter strain and the age of the cheese used for the extract. The decrease in the counts of Lb. plantarum I91 was observed mainly in the extracts in which the pH had diminished the most. In addition, the extracts that best supported the viability of Lb. plantarum I91 during incubation had the highest free amino acids content. The effect of Lb. plantarum I91 on the proteolysis profile of the extracts was marginal. Significant changes in the content of free amino acids suggested that the catabolism of free amino acids by Lb. plantarum I91 prevailed in a weakly proteolyzed medium, whereas the release of amino acids due to peptidolysis overcame their catabolism in a medium with high levels of free amino acids. Lactobacillus plantarum I91 was able to use energy sources other than lactose to support its growth because equivalent numbers of cells were observed in extracts containing residual amounts of lactose and in lactose-depleted extracts. The contribution of Lb. plantarum I91 to hard-cooked cheese peptidolysis was negligible compared with that of the starter strain; however, its ability to transform amino acids is a promising feature of this strain.

Two strains of nonstarter lactobacilli increased the production of flavor compounds in soft cheeses.

The contribution to flavor generation and secondary proteolysis of 2 strains of mesophilic lactobacilli isolated from cheese was studied. Miniature soft cheeses (200 g) were produced with or without the inclusion of a culture of Lactobacillus plantarum I91 or Lactobacillus casei I90 in the starter composed of Streptococcus thermophilus. During ripening, cheeses containing the added lactobacilli showed an increased content of total free amino acids, but this increase was only significant in cheeses with Lb. plantarum I91. In addition, free amino acid profiles were modified by selective increases of some amino acids, such as Asp, Ser, Arg, Leu, and Phe. Cheeses inoculated with Lb. plantarum I91 or Lb. casei I90 were also characterized by a significantly higher concentration of diacetyl, a key flavor compound, and an increased content of acetoin. Results suggest an increase in the catabolism of either citrate or aspartate, with the production of the derived aroma compounds. Overall, aspartate content increased in both lactobacilli-added cheeses, whereas citrate was more or less constant, suggesting that aspartate could be the source of increased diacetyl and acetoin. A triangle aroma test showed that the addition of the lactobacilli strains significantly changed the sensory attributes of cheeses. At least 11 of 12 panelists commented that the aroma of cheeses with adjuncts was more buttery than that of control cheeses, which is desirable in most soft cheeses. Both Lb. plantarum I91 and Lb. casei I90 performed well as adjunct cultures by influencing cheese aroma development and cheese proteolysis.

Impact of chymosin- and plasmin-mediated primary proteolysis on the growth and biochemical activities of lactobacilli in miniature Cheddar-type cheeses.

Strongly proteolytic starters seem to improve the growth of nonstarter lactobacilli during cheese ripening, but no information is available on the impact of the nonmicrobial proteases usually active in cheese on their development. In the current study, the influence of chymosin- and plasmin-mediated proteolysis on the growth and biochemical activities of lactobacilli during ripening of miniature Cheddar-type cheeses, manufactured under controlled microbiological conditions, was studied. Two experiments were performed; in the first, residual chymosin activity was inhibited by the addition of pepstatin, and in the second, plasmin activity was increased by adding more enzyme, obtained in vitro through the activation of plasminogen induced by urokinase. Cheeses with or without a Lactobacillus plantarum I91 adjunct culture and with or without added pepstatin or plasmin solution were manufactured and ripened for 60 d. The addition of the adjunct culture resulted in enhancement of secondary proteolysis, evidenced by an increase in the total content of free amino acids (FAA) and modifications of the individual FAA profiles. Reduction in residual chymosin activity caused a decrease in primary and secondary proteolysis, characterized by the absence of alpha(s1)-casein hydrolysis and reduced production of peptides and FAA, respectively. The increase in plasmin activity accelerated primary proteolysis but no enhancement of secondary proteolysis was observed. Chymosin- and plasmin-mediated proteolysis did not influence the growth and biochemical activities of adventitious or adjunct lactobacilli, indicating that it is not a limiting factor for the development and proteolytic-peptidolytic activities of lactobacilli in the cheese model studied.

Viability and contribution to proteolysis of an adjunct culture of Lactobacillus plantarum in two model cheese systems: cheddar cheese-type and soft-cheese type.

AIMS: The influence of the cheese-making process, ripening conditions and primary starter on the viability and proteolytic activity of an adjunct culture of Lactobacillus plantarum I91 was assessed in two miniature cheese models, representative of Cremoso Argentino and Cheddar cheeses. METHODS AND RESULTS: Cheeses with and without adjunct culture were made under controlled microbiological conditions and sampled during ripening for physicochemical and microbiological analyses. The addition of lactobacilli neither contributed to acid production nor caused changes to the composition of the cheeses. The strain studied exhibited good development and survival and showed a similar growth pattern in both cheese matrices. The adjunct culture caused changes to secondary proteolysis of both cheese types, which were evidenced by modification of peptide profiles and the increase in the levels of some individual amino acids as well as the total content of free amino acids. The changes observed were consistent with the acceleration of proteolysis in the two cheese models assayed. CONCLUSION: Lactobacillus plantarum I91 has desirable and robust technological properties, which makes it a suitable adjunct culture for cheese-making. SIGNIFICANCE AND IMPACT OF THE STUDY: Other cultures and environmental conditions prevailing in the food may affect the viability of adjunct cultures and its biochemical activities; this is the first report describing the successful performance of an adjunct culture of Lact. plantarum I91 in two different model cheese systems.

Endometrial ablation with a vaporizing electrode. I. Evaluation of in vivo effects.

BACKGROUND: To evaluate histologically and histochemically the physical and thermal effects of a vaporizing electrode as compared with a standard cutting loop in the performance of endometrial ablation. METHODS: Operative hysteroscopy was performed on 20 menorrhagic patients immediately before hysterectomy. Part of the posterior uterine wall was treated with a cylindrical, grooved, vaporizing electrode and undamped current set at 200 watts, and part with a standard cutting loop and undamped current set at 100 watts. A mucosal strip of about 1 cm width was left intact between the two treatment areas. Specimens underwent histologic examination after hematoxylin and eosin staining and histochemical assessment of thermal injury was based on detection of the respiratory enzyme dihydronicotinamide adenine dinucleotide diaphorase. RESULTS: The mean (standard deviation) endometrial thickness as determined on the untreated area of the posterior uterine wall was 1.08 (0.36) mm. The mean depth of furrows was 3.10 (0.90) mm with the use of the vaporizing electrode and 3.41 (1.11) mm after passage of the cutting loop. Corresponding values when thermal necrosis beneath the ablated area was assessed by the dihydronicotinamide adenine dinucleotide diaphorase technique were, respectively, 1.80 (0.40) mm and 0.41 (0.20) mm (mean difference, 1.39 mm; 95% confidence interval, 1.19 to 1.59: p<0.001, Mann-Whitney U test). CONCLUSIONS: A vaporizing electrode and a standard cutting loop obtained a similar degree of endomyometrial ablation. However, the depth of the thermal effect of the former electrode was significantly greater. Clinical studies are warranted, also considering the potential limitation of fluid absorption and menorrhagia recurrence when a vaporizing electrode is used.

Endometrial ablation with a vaporizing electrode. II. Clinical outcome of a pilot study.

BACKGROUND: As a vaporizing electrode has been successfully used to treat submucous myomas, we evaluated its safety and efficacy in performing endometrial ablation. METHODS: Forty consecutive women with established menorrhagia with (n=26) and without (n=14) submucous myomas were enrolled in a prospective, noncomparative, pilot study. Hysteroscopic endometrial vaporization was performed with pure cutting current set at 200 watts. RESULTS: All procedures were completed without complications. Median (interquartile range, IQR) fluid deficit was 90 (0-200) ml and median (IQR) operating time 10 (7-12.5) min. A significant correlation was observed between operating time and fluid absorption (Spearman's test by ranks, r=0.47; p=0.002). The degree of difficulty of the operation was classified as none on 28 (70%) occasions, mild on 11 (27.5%) and moderate on one (2.5%). After a mean+/-s.d. follow-up of 20.3+/-2.4 months, amenorrhea or spotting were reported by 23 (57.5%) subjects, hypomenorrhea by 10 (25%), normal flows by six (15%), and menorrhagia by one (2.5%). The median (IQR) menstrual score calculated according to a pictorial blood loss assessment chart dropped from 282.5 (199-383) to 0 (0-15) (p<0.0001). Six (15%) subjects were very satisfied with the effect of surgery, 30 (75%) satisfied, two (5%) uncertain, one (2.5%) dissatisfied and one (2.5%) very dissatisfied. CONCLUSIONS: The vaporizing electrode seems to combine the benefits of the cutting loop (speed, efficacy and possibility of removing myomas) and the roller-ball electrode (safety and limited fluid absorption) while avoiding their respective disadvantages, and may be considered an interesting alternative in the hysteroscopic treatment of menorrhagia.

[The diagnosis and therapy of HPV-associated genital lesions: the role of systemic beta-interferon treatment]. / Diagnostica e terapia delle lesioni genitali associate ad HPV: ruolo del trattamento sistemico con beta-interferone.

OBJECTIVE: To evaluate the effective role of HPV DNA typing by commercial probes and of i.m. interferon therapy in the management of HPV-related female genital lesions. EXPERIMENTAL DESIGN: Perspective study in a second-level colposcopy service (Istituto Ostetrico-Ginecologico "L. Mangiagalli"--II Clinica Ostetrica-Ginecologica). 110 patients, age range 15-45 years, with cytologic and/or histologic diagnosis of HPV-related genital lesions: HPV DNA typing (commercial probes) of genital specimens was performed. Excluding CIN III or VIN III cases, patients were treated with i.m. interferon-beta therapy for 4 weeks and, in case of persistence, for other 4 weeks. A colposcopic, cytologic and histologic follow-up for 6 months after therapy was performed. HPV DNA typing was repeated only in case of positive hybridization before therapy. RESULTS: In 81 cases, diagnosis of low-grade intraepithelial genital lesion (LSIL) was done, while 29 were high-grade intraepithelial genital lesions (HSIL). 73 patients completed the study. 3 months after interferon-beta i.m. administration, we observed complete remission in 72.97% of LSILs and in 47.36% of HSILs, while, 6 months after, remissions were observed in 81.25% of LSILs and in 73.33% of HSILs. Anyhow, treatment was well tolerated. HPV DNA typing confirmed the clinical course of lesions. CONCLUSIONS: Because of difficulties to standardize techniques, we believe that HPV DNA typing cannot assume a clinical or prognostic value, but only an experimental one. Interferon-beta i.m. administration could be considered a valid alternative or support to physical therapy as treatment of HPV-related genital lesions.

[Certain aspects of the relationship between the patient with a heart transplant and the donor's family]. / Indagine sugli aspetti relazionali tra il soggetto trapiantato di cuore e la famiglia del donatore.

The anonimity of cardiac transplant donors and recipients is protected by law but very often media and the press disclose information that are supposed to be strictly confidential. Aim of the survey was to explore how many cardiac transplant recipients are aware of the identity of the donors, if they had any kind of contact with the family of the donor and their overall opinion on the law that protects anonymity. All the patients who underwent a cardiac transplant in the Bergamo Hospital from 1985 to 1991 were mailed a questionnaire. 152 of 154 questionnaires were returned (two patients had died). Only a minority of subjects 44 (29%) contacted the family of the donor and 36 (86.3%) were still in touch. 50% of the subjects knew the name of the donor; 67% of patients would disclose the information of the identity of the donor only if specifically requested by both parties. The results of the survey stress the importance of the issue of the cardiac transplant patients information, both for the patient and the staff.

Automatic search for maximum similarity between molecular electrostatic potential distributions.

A new computer program has been developed to automatically obtain the relative position of two molecules in which the similarity between molecular electrostatic-potential distributions is greatest. These distributions are considered in a volume around the molecules, and the similarity is measured by the Spearman rank coefficient. The program has been tested using several pairs of molecules: water vs. water; phenylethylamine and phenylpropylamine vs. benzylamine; and methotrexate vs. dihydrofolic acid.

[High-grade sarcoma with areas of mullerian adenosarcoma of the uterus]. / Sarcoma ad alto grado con aree di adenosarcoma mulleriano dell'utero.

This paper describes a case of mullerian adenosarcoma with sarcomatous overgrowth. This 75-year old woman presented with atypical vaginal bleeding and enlarged uterus. On ultrasonography the endometrium was described as "thick" (4 cm) with cysts and aggregates of sarcomatous cells were seen on VABRA curettage. TAH & BSO was performed. The polypoid intrauterine mass measured 10 cm in the largest size and protruded through the external orifice. It was yellow with scattered cysts at the base and hemorrhagic at the apex. Myometrial invasion was evident. Microscopically most of the tumor consisted of a fibrosarcoma like tissue. It contained rhabdomyoblastic elements and mitosis count was 15 or more for 10 HPF. A small part of the tumor shared the features of mullerian adenosarcoma with glands surrounded by condensed stroma. The pseudostratified glandular epithelium was ciliated and focally mucinous. Periglandular tissue was pleiomorphic and mitotically active. Immunohistochemically stromal cells showed a slight positive staining for vimentin and a strong positivity for smooth muscle specific alpha actin. With the exception of scattered cells, desmin and striated muscle specific actin were extensively negative.

Kinetic pattern of human urinary transitional cell carcinoma.

The potential proliferative activity, expressed as labelling index, has been studied in 24 cases of bladder transitional cell carcinomas. The data showed a wide range of values, varying between 0.4 and 36%. The average proliferative activity was 3.2%. An analysis of the connection between the labelling index and the histological grading has also brought out the existence of a gradient of proliferative activity; average values of 1.6-2.53% and 3.49%, respectively, were found for grades I-II and grade III.