High throughput phenotyping of cross-sectional morphology to assess stalk lodging resistance

Background Stalk lodging (mechanical failure of plant stems during windstorms) leads to global yield losses in cereal crops estimated to range from 5% to 25% annually. The cross-sectional morphology of plant stalks is a key determinant of stalk lodging resistance. However, previously developed techniques for quantifying cross-sectional morphology of plant stalks are relatively low-throughput, expensive and often require specialized equipment and expertise. There is need for a simple and cost-effective technique to quantify plant traits related to stalk lodging resistance in a high-throughput manner. Results A new phenotyping methodology was developed and applied to a range of plant samples including, maize (Zea mays), sorghum (Sorghum bicolor), wheat (Triticum aestivum), poison hemlock (Conium maculatum), and Arabidopsis (Arabis thaliana). The major diameter, minor diameter, rind thickness and number of vascular bundles were quantified for each of these plant types. Linear correlation analyses demonstrated strong agreement between the newly developed method and more time-consuming manual techniques (R2 > 0.9). In addition, the new method was used to generate several specimen-specific finite element models of plant stalks. All the models compiled without issue and were successfully imported into finite element software for analysis. All the models demonstrated reasonable and stable solutions when subjected to realistic applied loads. Conclusions A rapid, low-cost, and user-friendly phenotyping methodology was developed to quantify two-dimensional plant cross-sections. The methodology offers reduced sample preparation time and cost as compared to previously developed techniques. The new methodology employs a stereoscope and a semi-automated image processing algorithm. The algorithm can be used to produce specimen-specific, dimensionally accurate computational models (including finite element models) of plant stalks.

Calcarea carbonica derivative complex (M8) as adjuvant treatment of inflammatory mammary carcinoma in a dog

Background: Inflammatory mammary carcinoma (IMC) is locally aggressive, fast growing, highly malignant tumor that affects humans and dogs. Affected dogs usually are presented with generalized edema, pain, erythema, and skin ulceration in mammary glands. Surgery is not recommended and an effective treatment has not been established [1]. Calcarea carbonica derivative complex (M8) has demonstrated anticancer properties in a murine model, by improving innate immune response against tumor cells [2,3]. M8 is a complex high diluted medication comprised of a 10%-20% concentration of Calcarea carbonica, Aconitum napellus, Arsenicum album, Asa foetida, Conium maculatum, Ipecacuanha, Phosphorus, Rhus tox, Silicea, Sulphur, and Thuya occidentalis, all in decimal dilutions of Hahnemann in distilled water and submitted to vigorous shaking. Aim: Describe an association of M8 and piroxicam (Non-steroidal anti-inflammatory drug) to treat a dog with IMC. Discussion: A 7 years old, mixed breed intact female dog was presented to the Federal University of Parana - Veterinary Hospital, Curitiba (HV-UFPR) for mammary glands examination. The owners related inflammation of mammary glands with clinical course of approximately 10 days, which was treated for mastitis (cephalexin and metergoline) without clinical improvement. Clinical examination revealed erythema, increased skin warmth, pain on palpation, and plaque involving the 4th and 5th right mammary glands. Abdominal ultrasound and serum biochemistry were unremarkable. Thoracic radiographs showed suspicious images of pulmonary metastasis. Fine needle biopsy was taken for cytologic examination. Cytological interpretation was a malignant epithelial neoplasm, probably a mammary carcinoma. Diagnosis of IMC was based on clinical signs and cytopathology. Dog was treated with oral (0.5 mL) and topical M8 twice a day for 15 days, and pyroxican, 0.3mg/kg, PO, q24h. Clinical improvement was observed 7 days after starting treatment. Until present date (70 treatment days with M8), dog has no clinical signs of IMC, and does not show signs of disease progression. Conclusion: The present report suggests that M8 associated with piroxicam contributes to improvement of IMC dog’s quality of life and survival rate. However, further clinical studies are needed to evaluate response to treatment in patients diagnosed with IMC.

In vivo treatment with M8, a highly diluted tinctures complex, reduced the malignancy of a mouse melanoma model

Background: Cancer is a class of disease responsible for 13% of death cause worldwide. Among all types of cancers, one of the most aggressive and with the highest death rate is melanoma. It is highly metastatic and current treatments with chemotherapeutic drugs do not yield satisfactory results. Therefore, the interest on new therapeutics for cancer treatment has been increasing on research. Highly diluted tinctures (HDT) are intended to enhance immune system responses resulting in reduced frequency of various diseases, and often present no risk of serious side-effects due to its low toxicity. Previous results have demonstrated in vitro inhibition of invasion ability and in vivo anti-metastatic potential of B16F10 lung metastasis model after mice treatment with M8 inhalation. Aims: Now we have evaluated M8 effects on hyaluronic acid and its specific melanoma cell surface receptor (CD44) expression on lungs after inhalation by mice. Methodology: M8 compounds include Aconitum napellus 20dH, Arsenicum album 18dH, Asa foetida 20dH, Calcarea carbonica 16dH, Conium maculatum 17dH, Ipecacuanha 13dH, Phosphorus 20dH, Rhus toxicodendron 17H, Silicea 20dH, Sulphur 24dH, and Thuja occidentalis 19dH. B16F10 Melanoma cells were inoculated into C57B/L6 mouse lateral tail vein. Treatment started 24 hours after inoculation, and was repeated after each 12 hours during 14 days on an inhalation chamber that is adapted to little rodents. Mice were subjected to euthanasia by intraperitoneal injection of thiopental followed by decapitation. Lungs were surgically removed and analyzed under a stereomicroscope for the presence of metastatic foci. They were formaldehyde fixed, dehydrated and paraffin embedded. Histological sections were processed for hematoxilin/eosin (HE), Fontana-Masson and immunohistochemistry staining methods. Images were captured and blindly analysed by ImageJ (NIH) software. Results: HE and Fontana-Masson showed a reduction in number and size of metastatic nodules, as previously demonstrated. We have detected a reduction on hyaluronic acid as well as CD44 expression on mice lungs after M8 treatment. The high metastatic potential of melanoma is proportional to hyaluronic acid expression level, together with its specific cell surface receptor, the CD44. These results suggest that M8 treatment reduces malignancy of mouse melanoma through modulation of hyaluronic acid and CD44 expression, which play crucial roles in tumor invasion and growth. Conclusion: Even though further investigation are necessary to elucidate the mechanisms of action of M8 treatment there is an indication that these highly diluted tinctures could be a promising therapy to treat metastatic melanoma.

Causticum hahnemanni, Conium maculatum and Lycopodium clavatum highly diluted medications decreases parasitemia in mice infected by Trypanosoma cruzi

Introduction: Benznidazole is the only medicine available in Brazil for Chagas’ disease treatment, however it presents low efficacy in the chronic phase and several adverse effects [1-3]. Aim: Evaluate the effect of Causticum hahnemanni, Conium maculatum and Lycopodium clavatum [4-6] administered to mice infected with T. cruzi. Method: In blind randomized controlled trial 42 male Swiss mice, 8 weeks of age, have been grouped: GCaus –treated with C. hahnemanni 13cH (n=10), GCon –treated with C. maculatum 13 cH (n=11), GLy –treated with L. clavatum13cH (n=10) and CG – control group (n=11) treated with 7% hydro alcoholic solution 13cH. The animals were infected intraperitoneally with 1.400 blood trypomastigotes of T. cruzi - Y strain. Medications were been prepared according to Brazilian Homeopathic Pharmacopoeia [7]. Medication was diluted in water (1mL/100mL) offered ad libitum, from amber recipient during 16 hours administered 48 hours before infection and 48, 96 and 144 hours after inoculation. Parasitological parameters assessed: total parasitemia (TP), maximum peak of parasites (MPP), pre-patent period (PPP) and area under curve (AUC). Parasitemia was evaluated daily counting from the first day of infection. Clinical parameters assessed: weight, temperature, water and food intake and excreta were measured counting from the 5º day before infection until animals’ death or checking negative parasitemia for 3 consecutive days. Mortality was registered for 75 days after infection. Ethics Committee for Experiments in Animals gave approval UEM 054/11. Statistical comparison of data was performed with Kruskal-Wallis test, with 5% significance. Results and Discussion: The diluted medications have been significantly reduced the parasitological parameters: MPP (p

Oral, topical, and inhalation of Calcarea carbonica derivative complex (M8) to treat inflammatory mammary carcinoma in dogs

Background: Inflammatory mammary carcinoma (IMC) is locally aggressive, fast growing, highly malignant tumor that affects humans and dogs. Affected dogs usually are presented with generalized edema, pain, erythema, and skin ulceration in mammary glands. Surgery is not recommended and an effective treatment has not been established [1]. Calcarea carbonica derivative complex (M8) has demonstrated anticancer properties in a murine model, by improving innate immune response against tumor cells [2]. M8 is a complex high diluted medication comprised of Calcarea carbonica 16x, Aconitum napellus 20x, Arsenicum album 18x, Asa foetida 20x, Conium maculatum 17x, Ipecacuanha 13x, Phosphorus 20x, Rhus toxicodendron 17x, Silicea 20x, Sulphur 24x, and Thuya occidentalis 19x, dilution procedures have followed standard methodology described at the Brazilian Homeopathic Pharmacopoeia. Aims: To describe different routes of M8 administration associated with oral pyroxican (non-steroidal anti-inflammatory drug) to treat dogs with IMC. Methodology: Three female dogs with 10 years old median age were presented to the Veterinary Teaching Hospital at Federal University of Parana, Curitiba (HV-UFPR) with cytological and clinical diagnosis of IMC. Patients were treated with oral (0.5 mL,q12h), topical (q12h) and inhalatory (2 mL, q24h, through an ultrasonic inhalation device) M8, and oral pyroxican (0.3mg/kg, q24h).Thoracic radiographs showed pulmonary metastasis in all dogs. Results: 7 days after initiating treatment all patients had clinical improvement. It was observed reduction on mammary glands inflammation and decreased pain sensitivity. One patient had 8 month of complete remission. The other two patients died 1 and 2 month after initial treatment. However none of the patients had pulmonary progressive disease, showed by radiographic examinations. Owners revealed treatment satisfaction in regards to quality of life improvement, easy M8 administration, good M8 palatability for dogs, and inflammation reduction. Conclusion: The present report suggests that M8 influenced positively the anti -inflammatory treatment. Keywords: Calcarea carbonica complex; inflammatory mammary carcinoma; routes of administration References [1] Sorenmo K. Canine mammary gland tumors. Veterinary Clinics of North America: Small Animal Practice. 2003 33(3):573-96. [2] Oliveira CC, Abud APR, Oliveira SM, Guimarães FSF, Andrade LF, Di Bernardi RP, Coletto ELO, Kuczera D, Da Lozzo EJ, Gonçalves JP, Trindade ES, Buchi DF. Developments on drug discovery and on new therapeutics: highly diluted tinctures act as biological response modifiers. BMC Complementary and Alternative Medicine 2011, 11(101): 2-11.

Experimental induced wound cicatrisation after highly diluted products treatment

Introduction: Skin is an attractive target to study extracellular matrix, due to abundance in Connective tissue. In cases of injuries the first step is an inflammatory reaction and subsequent the healing that involves several changes in the matrix. These changes are fundamental to inflammatory cells activities allowing healing. Highly diluted products were shown to facilitate inflammatory mediators and to activate immune cells in vivo and in vitro, thus it can be effective to wound healing. Aims: This study aims to evaluate highly diluted products effects on inflammation and cicatrization process. Methodology: Three compounds (M8 (Aconitum napellus 20dH, Arsenicum album 18dH, Asa foetida 20dH, Calcarea carbonica 16dH, Conium maculatum 17dH, Ipecacuanha 13dH, Phosphorus 20dH, Rhus toxicodendron 17dH, Silicea 20dH, Sulphur 24dH, Thuja occidentalis 19dH), M1 (Chelidonium majus 20dH, Cinnamon 20dH, Echinaceae purpurea 20dH, Gelsemium sempervirens 20dH plus all M8 compounds) and Curcuma cH30 – simple product), were manipulated as a gel and applied on mice dorsal flank after incision and suture (approximately 1 cm and three points), for 3 consecutive days. After the treatments the scars were evaluated macroscopically, the animals were killed, the skin samples collected, fixed and processed for Hematoxilin-Eosin (HE) and Masson Tricromic (to observe the collagen fibers type I). The slices were analyzed and images collected by a light microscope Olympus BX51 with camera attached Olympus DP72. Results: It was observed a higher and faster rate of tissue epithelization in the treated groups after three days of gel-product application. This could be observed in lower rates in the control group (no treatment) - Figure 1 and 2). Regeneration and organization of connective tissue were proportional to epithelization the treated groups. We also observed evidences of changes in amount of neutrophils and fibroblasts, resulting in changes in the healing period. Analyses for these confirmations are in progress.

Evaluation of Calcarea carbonica derivative complex (M8) on milk parameters in the dairy cow

Background Any dairy herd that continually has a somatic cell count (SCC) above 200,000 cells/ml has an indication of mammary gland inflammation (mastitis). Routine use of antibiotics to prevent mastitis is prohibited by organic farming regulations. This limitation has lead researchers to focus on cows natural defense mechanisms [1]. Calcarea carbonica derivative complex (M8) is a complex high diluted medication comprised of comprised of Calcarea carbonica 16x, Aconitum napellus 20x, Arsenicum album 18x, Asa foetida 20x, Conium maculatum 17x, Ipecacuanha 13x, Phosphorus 20x, Rhus toxicodendron 17x, Silicea 20x, Sulphur 24x, and Thuya occidentalis 19x. Dilution procedures have followed standard methodology described at the Brazilian Homeopathic Pharmacopoeia. This medication has enhanced immune system responses both in vitro and in vivo in a murine model [2]. Aims In the present study, we investigate the response of dairy cows after M8 treatment. Methodology The study was performed as a randomized, observer double-blinded and placebo-controlled trial, with a stratified design, using lactation number and SCC as stratification factors. The study sample consisted of 42 lactating dairy cows (Holstein) in one high producing dairy herd with 52 cows in milk in southern Brazil, divided into two experimental groups (n=21). Exclusion criteria were cows with clinical mastitis or receiving any other medical treatment. Pre- and post-milking teat disinfection was practiced in the herd. All cows were clinically examined, with udder and milk samples being appraised according to Rosenberger (1990) [3]. During 3 months one group received daily M8 treatment, the other placebo. Oral administration of 5 ml/day/cow was performed using an automatic dosage dispenser. Monthly, milk production, SCC, fat and total protein content were carefully recorded for each animal by an official milk recording program. SCC were log transformed for analysis. ANOVA and Tukey test were used to compare the averages. The Bartlett´s test was used for homogeneity of variance evaluation. Results There were no significant differences (P=0.435) among the groups in the initial evaluation (Values of SCC x103 : Placebo 67,37±80,48; Treatment 359,39±677,02). After 3 months, the M8 treated group showed a decrease (134,00±178,76 P= 0.047) in SCC when compared with control group (391,71±686,60). Fat and protein did not differ between groups and time analysed. Milk production decreased in the placebo group during time (Before: 34,97±6,69kg; After:28,69±4,33kg), whereas the treatment group did not change total amount (Before: 28,7±6,54kg; After: 26,39±5,73kg; P > 0.05). Conclusions These results indicate that the M8 influenced positively SCC and suggest that it may be considered as a possible tool to promote bovine mastitis prophylaxis. Keywords: Calcarea carbonica complex, mastitis, somatic cell count [1] McDougall S, Parker KI, Heuer C, Compton CWR. A review of prevention and control of heifer mastitis via non-antibiotic strategies. Vet Microbiol. 2009; 154:177–85. [2] Oliveira CC, Abud APR, Oliveira SM, Guimarães FSF, Andrade LF, Di Bernardi RP, Coletto ELO, Kuczera D, Da Lozzo EJ, Gonçalves JP, Trindade ES, Buchi DF. Developments on drug discovery and on new therapeutics: highly diluted tinctures act as biological response modifiers. BMC Complementary and Alternative Medicine. 2011; 11(101): 2-11. [3]Rosenberger, G. Die klinische Untersuchung des Rindes, 3. Neubearbeitete Auflage, Paul Parey Verlag, Berlin, 1990.

Pilot Study: Evaluation of the efficacy of homeopathic anti-tumor protocols in Ehrlich tumor

Cancer is the second leading cause of death worldwide, with breast cancer one of the tumors with the highest incidence in Brazil, with an estimated 57,120 new cases in 2014. Several forms of treatment and control of cancer symptoms has been studied, among them homeopathy. In the Banerji clinic in India, doctors have used a protocol consisting of 4 homeopathic medicines with relevant results that motivated this study. In our research the Ehrlich tumor was used, a transplantable tumor cell, whose features are similar a breast carcinoma, as discovered in 1905 by Paul Ehrlich. Objective: To analyze the clinical and immunomodulatory Ehrlich tumor in mice treated with the homeopathic medications found in the Banerji Protocol and with thymulin 5CH. Methodology: The project was approved under protocol 158/13 of the Paulista University Ethics Committee. BALB / c mice between 6 and 9 weeks old were used. Each mouse was inoculated with 1x10 7 tumor cells into the peritoneum cavity, to obtain the ascitic form of tumor. Mice were divided in 5 groups (N=6 animals per group), being the following treatments: control treated with sterile water, Conium maculatum 200CH, Thuja occidentalis 200CH, Phytolacca decandra 200CH, Carcinosinum 200CH, every day being 0.1mL/10g body weight. The animals were weighed and the consumption of water and feed was measured daily. Clinical signals and body temperature were also registered daily. The survival of each group was also analyzed. Results: All groups presented reduction of body temperature one day before death. The peak of clinical symptoms in the majority of groups was in the D18, while Carcinosinum treated group had its peak in D29. The observed clinical symptoms were apathy, prostration, cyanosis, priapism, tachydyspnea, genital edema and secretion, decreased general activity, hair bristling and trembling. The control group had loose stools as unique symptom, seen in 20% of mice; the Carcinosinum treated group showed trembling as the sole symptom, that affected 20% of mice. The group treated with Conium maculatum showed higher incidence of clinical symptoms, involving up to 80% of mice, while the group treated with Carcinosinum showed lower incidence of clinical symptoms, affecting no more than 40% of mice, with weight reduction (reduction of ascites) and longer survival, when compared to the other groups. Conclusion: The results of the pilot study directed us to the second stage of the research, in which the histopathological and immunohistochemical observation will be assessed, being Carcinosinum the treatment chosen to this next step.

CONİUM MACULATUM L. – LƏKƏLİ BADYAN BİTKİSİNİN BİOLOJİ XÜSUSİYYƏTLƏRİ

Açar sözlər: Apiaceae Lindl., Conium maculatum L., zəhərli bitkilər, alkoloidlər, qlikozidlər Key words: Apiaceae Lindl., Conium maculatum L., poisonous plants, alkaloids, glycosides Hesablamalara görə Yer kürəsində təqribən 10 mindən çox zəhərli bitki növünün olduğu güman edilir ki, onların da böyük əksəriyyəti tropik və subtropik zonalarda yayılmışdır, lakin soyuq iqlim qurşaqlarında da zəhərli bitkilərə rast gəlinir. Bitkilərdə həyat fəaliyyətləri dövründə əmələ gələn və özündə toplanan maddələrdən bir çoxu insanlar və digər canlılar tərəfindən istifadə edilir. Ən çox şəkərli, vitaminli və digər bioaktiv maddələrin toplandığı bitkilər təbiətdə canlılar tərəfindən sərf edilir, lakin bəzi bitkilər də vardır ki, onların sintez edərək vegetativ və ya generativ orqanlarında topladıqları maddələr insan və ya heyvanların qidalanması zamanı onları zəhərləyir.