The Species-Specific Acquisition and Diversification of a K1-like Family of Killer Toxins in Budding Yeasts of the Saccharomycotina.

Killer toxins are extracellular antifungal proteins that are produced by a wide variety of fungi, including Saccharomyces yeasts. Although many Saccharomyces killer toxins have been previously identified, their evolutionary origins remain uncertain given that many of these genes have been mobilized by double-stranded RNA (dsRNA) viruses. A survey of yeasts from the Saccharomyces genus has identified a novel killer toxin with a unique spectrum of activity produced by Saccharomyces paradoxus. The expression of this killer toxin is associated with the presence of a dsRNA totivirus and a satellite dsRNA. Genetic sequencing of the satellite dsRNA confirmed that it encodes a killer toxin with homology to the canonical ionophoric K1 toxin from Saccharomyces cerevisiae and has been named K1-like (K1L). Genomic homologs of K1L were identified in six non-Saccharomyces yeast species of the Saccharomycotina subphylum, predominantly in subtelomeric regions of the genome. When ectopically expressed in S. cerevisiae from cloned cDNAs, both K1L and its homologs can inhibit the growth of competing yeast species, confirming the discovery of a family of biologically active K1-like killer toxins. The sporadic distribution of these genes supports their acquisition by horizontal gene transfer followed by diversification. The phylogenetic relationship between K1L and its genomic homologs suggests a common ancestry and gene flow via dsRNAs and DNAs across taxonomic divisions. This appears to enable the acquisition of a diverse arsenal of killer toxins by different yeast species for potential use in niche competition.

Increasing Appropriate BRCA1/2 Mutation Testing: The Role of Family History Documentation and Genetic Counseling in a Multidisciplinary Clinic.

BACKGROUND: Findings show that 5-10 % of women with a diagnosis of breast cancer (BCa) have actionable genetic mutations. The National Comprehensive Cancer Network guidelines for testing to detect BRCA1/2 mutations include personal history (PH) variables such as age of 45 years or younger and a family history (FH) variables. Rates of FH documentation and overall rates of appropriate referral for genetic testing are low, ranging from about 30 to 60 %. The authors hypothesized that an upfront FH documentation and inclusion of a genetics counselor in a multidisciplinary clinic (MDC) setting would increase rates of appropriate referral for genetic testing. METHODS: The study enrolled 609 consecutive women with non-metastatic BCa seen in consultation between June 2012 and December 2015 at a multidisciplinary clinic. Rates of FH documentation and referral for genetic testing to detect BRCA1/2 mutations were assessed before and after inclusion of a genetic counselor in the MDC. RESULTS: The rates of FH documentation and appropriate referral were 100 and 89 %, respectively. Half (50 %) of the patients had only FH-based indications for testing. All the patients with PH-based indications were referred. The inclusion of a genetic counselor significantly increased appropriate referral rates among those with only FH-based indications (62 vs. 92 %) and overall (80 vs. 96 %) (p < 0.0001 for both). Among the 12 % of the patients with actionable mutations, 60 % were 45 years of age or younger, whereas 30 % had only FH-based testing indications. CONCLUSIONS: This report shows substantially higher FH documentation and appropriate genetic testing rates than prior reports. Many patients with indications for genetic testing may have only FH-based indications for testing, and this subset may account for the sizable proportion of patients with newly diagnosed BCa who have actionable mutations.

Determining the genetic stability of micropropagated sugarcane using inter-simple sequence repeat markers.

Sugarcane culture is an important source of income for the Brazilian economy. The aim of this study was to identify somaclonal variation in sugarcane varieties RB943365 and RB92579 arising from micropropagation using inter-simple sequence repeat (ISSR) DNA markers. The evaluated plants were generated from the in vitro propagation of shoot tips grown in MS medium supplemented with vitamins, myoinositol, glycine, and sucrose, without the use of growth regulators. Fifteen consecutive subcultures with intervals of 14 days were carried out, and DNA was extracted from young leaves obtained from each of the subcultures. The DNA was amplified with ISSR markers and separated by electrophoresis on 2% agarose gels. No evidence of polymorphism was observed in subcultures of the varieties analyzed, suggesting the absence of somaclonal variants. In this way, the ISSR marker was efficient at analyzing somaclonal variation, and in vitro propagation of sugarcane can be considered efficient for 15 consecutive subcultures of the varieties analyzed.

A protective effect after clearance of orthotopic rat hepatocellular carcinoma by nanosecond pulsed electric fields.

Strategies for treating liver cancer using radiation, chemotherapy combinations and tyrosine kinase inhibitors targeting specific mutations have provided longer survival times, yet multiple treatments are often needed and recurrences with new malignant phenotypes are not uncommon. New and innovative treatments are undoubtedly needed to successfully treat liver cancer. Over the last decade, nanosecond pulsed electric fields (nsPEFs) have shown promise in pre-clinical studies; however, these have been limited to treatment of skin cancers or xenographs in mice. In the present report, an orthotopic hepatocellular carcinoma (HCC) model is established in rats using N1-S1 HCC cells. Data demonstrate a response rate of 80-90% when 1000 pulses are delivered with 100ns durations, electric field strengths of 50kV/cm and repetition rates of 1Hz. N1-S1 tumours treated with nsPEFs expressed significant number of cells with active caspase-3 and caspase-9, but not caspase-8, indicating an intrinsic apoptosis mechanism(s) as well as caspase-independent mechanisms. Most remarkably, rats with successfully ablated tumours failed to re-grow tumours when challenged with a second injection of N1-S1 cells when implanted in the same or different liver lobe that harboured the original tumour. Given this protective effect, infiltration of immune cells and the presence of granzyme B expressing cells within days of treatment suggest the possibility of an anti-tumour adaptive immune response. In conclusion, NsPEFs not only eliminate N1-S1 HCC tumours, but also may induce an immuno-protective effect that defends animals against recurrences of the same cancer.

Genetic variability and resistance of cultivars of cowpea [Vigna unguiculata (L.) Walp] to cowpea weevil (Callosobruchus maculatus Fabr.).

The cowpea weevil (Callosobruchus maculatus Fabr.) is the most destructive pest of the cowpea bean; it reduces seed quality. To control this pest, resistance testing combined with genetic analysis using molecular markers has been widely applied in research. Among the markers that show reliable results, the inter-simple sequence repeats (ISSRs) (microsatellites) are noteworthy. This study was performed to evaluate the resistance of 27 cultivars of cowpea bean to cowpea weevil. We tested the resistance related to the genetic variability of these cultivars using ISSR markers. To analyze the resistance of cultivars to weevil, a completely randomized test design with 4 replicates and 27 treatments was adopted. Five pairs of the insect were placed in 30 grains per replicate. Analysis of variance showed that the number of eggs and emerged insects were significantly different in the treatments, and the means were compared by statistical tests. The analysis of the large genetic variability in all cultivars resulted in the formation of different groups. The test of resistance showed that the cultivar Inhuma was the most sensitive to both number of eggs and number of emerged adults, while the TE96-290-12-G and MNC99-537-F4 (BRS Tumucumaque) cultivars were the least sensitive to the number of eggs and the number of emerged insects, respectively.

Unusual head metastasis of kidney cancer.

Renal cell carcinoma (RCC) metastasis can involve any organ. Head metastasis are rare, namely tongue, scalp and nose and only a few cases have been described. The authors describe three cases of unusual presentation of head metastasis of RCC. In case 1, a 50-year-old man with RCC underwent left nephrectomy, and presented 16 months later with nasal metastasis. In case 2, a 64-year-old woman with RCC operated 12 years ago, relapsed at multiple sites including scalp, which was treated surgically. In case 3, a 47-year-old man with RCC developed metastasis in multiple organs including a mass on the tongue that was operated and was compatible with primary tumour metastasis. These cases show the importance of correct diagnosis of rare presentations and local treatment of metastasis of RCC.

Induction of Cell Death Mechanisms and Apoptosis by Nanosecond Pulsed Electric Fields (nsPEFs).

Pulse power technology using nanosecond pulsed electric fields (nsPEFs) offers a new stimulus to modulate cell functions or induce cell death for cancer cell ablation. New data and a literature review demonstrate fundamental and basic cellular mechanisms when nsPEFs interact with cellular targets. NsPEFs supra-electroporate cells creating large numbers of nanopores in all cell membranes. While nsPEFs have multiple cellular targets, these studies show that nsPEF-induced dissipation of ΔΨm closely parallels deterioration in cell viability. Increases in intracellular Ca2+ alone were not sufficient for cell death; however, cell death depended of the presence of Ca2+. When both events occur, cell death ensues. Further, direct evidence supports the hypothesis that pulse rise-fall times or high frequency components of nsPEFs are important for decreasing ΔΨm and cell viability. Evidence indicates in Jurkat cells that cytochrome c release from mitochondria is caspase-independent indicating an absence of extrinsic apoptosis and that cell death can be caspase-dependent and -independent. The Ca2+ dependence of nsPEF-induced dissipation of ΔΨm suggests that nanoporation of inner mitochondria membranes is less likely and effects on a Ca2+-dependent protein(s) or the membrane in which it is embedded are more likely a target for nsPEF-induced cell death. The mitochondria permeability transition pore (mPTP) complex is a likely candidate. Data demonstrate that nsPEFs can bypass cancer mutations that evade apoptosis through mechanisms at either the DISC or the apoptosome.

Nanosecond pulsed electric fields (nsPEFs) activate intrinsic caspase-dependent and caspase-independent cell death in Jurkat cells.

NsPEF ablation induces apoptosis markers, but specific cell death pathways have not been fully defined. To identify nsPEF-activated cell death pathways, wildtype human Jurkat cells and clones with deficiencies in extrinsic and intrinsic apoptosis pathways were investigated. NsPEFs activated caspase isozymes and induced identical electric field-dependent cell death in clones deficient in FADD or caspase-8, indicating that extrinsic apoptosis pathways were not activated. This was confirmed when cytochrome c release was shown to be unaffected by the pan caspase inhibitor, z-VAD-fmk. NsPEF-treated APAF-1-silenced cells did not exhibit caspase-3/7 and -9 activities and corresponding electric field-dependent cell death in this clone was attenuated compared to its vector control at low, but not at high electric fields. These data demonstrate that nsPEFs induce intrinsic apoptosis activate by cytochrome c release from mitochondria through an APAF-1- and caspase-dependent pathway as well as through caspase-independent mechanisms that remain to be defined. Furthermore, the results establish that nsPEFs can overcome natural and oncogenic mechanisms that promote cell survival through inhibition of apoptosis and other cell death mechanisms.

Transient features in nanosecond pulsed electric fields differentially modulate mitochondria and viability.

It is hypothesized that high frequency components of nanosecond pulsed electric fields (nsPEFs), determined by transient pulse features, are important for maximizing electric field interactions with intracellular structures. For monopolar square wave pulses, these transient features are determined by the rapid rise and fall of the pulsed electric fields. To determine effects on mitochondria membranes and plasma membranes, N1-S1 hepatocellular carcinoma cells were exposed to single 600 ns pulses with varying electric fields (0-80 kV/cm) and short (15 ns) or long (150 ns) rise and fall times. Plasma membrane effects were evaluated using Fluo-4 to determine calcium influx, the only measurable source of increases in intracellular calcium. Mitochondria membrane effects were evaluated using tetramethylrhodamine ethyl ester (TMRE) to determine mitochondria membrane potentials (ΔΨm). Single pulses with short rise and fall times caused electric field-dependent increases in calcium influx, dissipation of ΔΨm and cell death. Pulses with long rise and fall times exhibited electric field-dependent increases in calcium influx, but diminished effects on dissipation of ΔΨm and viability. Results indicate that high frequency components have significant differential impact on mitochondria membranes, which determines cell death, but lesser variances on plasma membranes, which allows calcium influxes, a primary determinant for dissipation of ΔΨm and cell death.

Pathogenicity for onion and genetic diversity of isolates of the pathogenic fungus Colletotrichum gloeosporioides (Phyllachoraceae) from the State of Pernambuco, Brazil.

Onion anthracnose, caused by Colletotrichum gloeosporioides, is one of the main diseases of onions in the State of Pernambuco. We examined the pathogenicity of 15 C. gloeosporioides strains and analyzed their genetic variability using RAPDs and internal transcribed spacers (ITS) of the rDNA region. Ten of the strains were obtained from substrates and hosts other than onion, including chayote (Sechium edule), guava (Psidium guajava), pomegranate (Punica granatum), water from the Capibaribe River, maracock (Passiflora sp), coconut (Cocus nucifera), surinam cherry (Eugenia uniflora), and marine soil; five isolates came from onions collected from four different regions of the State of Pernambuco and one region of the State of Amazonas. Pathogenicity tests were carried out using onion leaves and bulbs. All strains were capable of causing disease in leaves, causing a variable degree of lesions on the leaves; four strains caused the most severe damage. In the onion bulb tests, only three of the above strains caused lesions. Seven primers of arbitrary sequences were used in the RAPD analysis, generating polymorphic bands that allowed the separation of the strains into three distinct groups. The amplification products generated with the primers ITS1 and ITS4 also showed polymorphism when digested with three restriction enzymes, DraI, HaeIII and MspI. Only the latter two demonstrated genetic variations among the strains. These two types of molecular markers were able to differentiate the strain from the State of Amazonas from those of the State of Pernambuco. However, there was no relationship between groups of strains, based on molecular markers, and degree of pathogenicity for onion leaves and bulbs.

Antinociceptive effects of the non-selective cannabinoid receptor agonist CP 55,940 are absent in CB1(-/-) and not CB2(-/-) mice in models of acute and persistent pain.

Previous studies have suggested a role for both CB1 and CB2 cannabinoid receptors in modulation of nociception. To further examine the role of CB1 and CB2 receptors in antinociception, we evaluated the efficacy of the non-selective cannabinoid receptor agonist, CP 55,940, in models of acute, inflammatory, and neuropathic pain in control mice, CB1 receptor knockout mice, and CB2 receptor knockout mice. In control C57BL/6 mice, administration of CP 55,940 (0.03-0.3 mg/kg, i.p.) reversed complete Freund's adjuvant-induced tactile allodynia, reversed tactile allodynia in the spinal nerve ligation model and inhibited the noxious heat-evoked tail withdrawal response. In addition to its antinociceptive effects, CP 55,940 produced an impairment of motor coordination in the rotarod test. The antinociceptive effects produced by CP 55,940 and associated motor deficits were found to be completely abolished in CB1 receptor knockout mice. In contrast, the antinociceptive effects of CP 55,940 in all pain models were fully retained in CB2 receptor knockout mice, along with the associated motor deficits. The results suggest that the antinociceptive effects of CP 55,940 in models of acute and persistent pain, along with the associated motor deficits, are mediated by CB1 receptors, and likely not CB2 receptors.

[Avandia and avandamet in the treatment of patients with diabetes mellitus type 2: effects on secretion of some fat tissue hormones].

AIM: To study effects of avandia and its combination with metformine (avandamet) on secretion of fat tissue hormones. MATERIAL AND METHODS: The examination protocol for 42 patients with type 2 diabetes mellitus (DM) aged 62.4 +/- 7.7 years comprised tests for blood levels of leptin, soluble receptor to leptin, insulin, grelin, resistin and adiponectine. RESULTS: The treatment resulted in reduction of fasting glycemia from 10.69 +/- 2.54 to 8.42 +/- 1.73 mmol/l, of glycosilated hemoglobin--from 8.1 + 1.6 to 7.75%, immunoreactive insulin--from 19.1 +/- 8.3 to 12.0 +/- 6.5 mg/ml, grelin--from 21.7 +/- 14.6 to 17.3 +/- 13.7 mg/ml, leptin--from 40.3 +/- 24.2 to 26.9 +/- 15.4 mg/ml, soluble receptor to leptin--from 17.9 +/- 4.5 to 13.1 +/- 3.5 mg/ml. LDLP and HDLP cholesterol was high. CONCLUSION: Avandia and avandamet are effective antidiabetic drugs with a beneficial effect on secretion of fat tissue hormones.

Cardiac amyloidosis in the cardiosurgical operating room--a rare but fatal trap.

Coronary artery bypass grafting resulted in a fatal outcome in two patients with unsuspected cardiac amyloidosis. Despite preoperative symptoms of myocardial ischemia, each case represented a different pathophysiological combination of cardiac amyloidosis and coronary artery disease. Pitfalls preventing correct diagnosis and possible patterns of treatment are discussed.

Evaluation of three scoring systems predicting non sentinel node metastasis in breast cancer patients with a positive sentinel node biopsy.

BACKGROUND: Completion axillary lymph node dissection (cALND), performed after a positive sentinel lymph node biopsy (SLNB) in breast cancer patients, often results in no additional positive nodes. Scoring systems have been published to aid in the prediction of nonsentinel node metastasis. Our purpose was to assess the validity of these scoring systems in our patient population. METHODS: For 39 consecutive patients who underwent cALND after a positive SLNB, scores were calculated using retrospective patient data for each of the three scoring systems used. Receiver operating characteristics (ROC) curves were drawn, and the areas under the curves were calculated to assess the discriminative power of each system. Univariate analysis was performed to assess the predictability of individual patient and tumor characteristics. RESULTS: Nonsentinel nodes were positive in 23 (59%) patients. The areas under the ROC curves were 0.63, 0.70, and 0.68, respectively. The proportion of sentinel nodes that were positive and the total number of sentinel nodes retrieved were the only individual predictors of nonsentinel node metastasis. CONCLUSIONS: Given the high incidence of retrieving no additional metastasis on cALND, individualized patient management according to risk is desirable. Scoring systems provide additional information regarding the likelihood of metastasis in nonsentinel nodes, but their predictability remains less than optimal. The use of scoring systems must be applied with caution until future studies provide a more accurate assessment of risk for patients with a positive SLNB.

Success of neoadjuvant chemotherapy in conversion of mastectomy to breast conservation surgery.

Neoadjuvant chemotherapy (NC) in patients with breast cancer results in high response rates and has been used with the purpose of reducing tumor size and achieving breast conservation (BC) in individuals who initially require mastectomy. Our objective is to determine the success of NC in achieving BC in women who initially were not candidates for BC. We conducted a cohort study of women with invasive breast cancer who required mastectomy but desired BC surgery. Outcomes measured were tumor response and rates of BC. Thirty-seven women had a mean age of 45 years. Mean tumor size was 51 mm, and 62 per cent were larger than 4 cm. Tumors were predominantly infiltrating ductal carcinoma (83.3%) and high grade (62.2%). Cyclophosphamide, doxorubicin, and 5-fluorouracil with or without taxotere were most commonly used (86%). Complete clinical and pathologic responses were seen in 32.4 per cent and 10.8 per cent of patients, respectively. BC was achieved in 56.7 per cent of cases. Only initial tumor size predicted tumor regression and success of BC (P = 0.014). Neither tumor histology nor biologic markers predicted tumor response. In conclusion, NC is an effective alternative in achieving tumor reduction and BC in selected patients who require mastectomy but desire BC surgery.

Intraoperative injection of technetium-99m sulfur colloid is effective in the detection of sentinel lymph nodes in breast cancer.

BACKGROUND: Our objective was to determine if intraoperative injection of technetium-99m-labeled sulfur colloid is as effective as preoperative injection in the detection of sentinel lymph nodes (SLNs). METHODS: Two hundred consecutive patients with breast cancer underwent SLN biopsy examination. Radiocolloid was injected in the preoperative area (group A) or immediately after induction of anesthesia in the operating room (group B). RESULTS: The SLN detection rate was similar for groups A (96%) and B (100%; P = .2). Radioactive SLNs were detected in 95% of patients in group A and in 97% of patients in group B (P = .1). The mean number of SLNs harvested was 1.6 and 2.1 for groups A and B, respectively. There was no significant difference in positive SLNs between groups (P = .11). CONCLUSIONS: Intraoperative injection of sulfur colloid is highly effective in the detection of SLNs, avoiding patient discomfort and surgical schedule delays.

Small bowel obstruction: a population-based appraisal.

BACKGROUND: Small bowel obstruction (SBO) is a common reason for surgical consultation, but little is known about the natural history of SBO. We performed a population-based analysis to evaluate SBO frequency, type of operation, and longterm outcomes. STUDY DESIGN: Using the California Inpatient File, we identified all patients admitted in 1997 with a diagnosis of SBO. Patients were excluded if they had a diagnosis of bowel obstruction in the previous 6 years (1991 to 1996). Of the remaining cohort, the natural history of SBO over the subsequent 5 years (1998 to 2002) was analyzed. Index hospitalization outcomes (eg, surgical versus nonsurgical management, length of stay, in-hospital mortality), and longterm outcomes, including SBO readmissions and 1-year mortality, were evaluated. RESULTS: We identified 32,583 patients with an index admission for SBO in 1997; 24% had surgery during the index admission. The distribution of surgical procedures was: 38% lysis of adhesions, 38% hernia repair, 18% small bowel resection with lysis of adhesions, and 6% small bowel resection with hernia repair. Patients who underwent operations during index admission had longer lengths of stay, lower mortality, fewer SBO readmissions, and longer time to readmission than patients treated nonsurgically. Regardless of treatment during the index admission, 81% of surviving patients had no additional SBO readmissions over the subsequent 5 years. CONCLUSIONS: Most of the 32,583 patients requiring admission for index SBO in 1997 were treated nonsurgically, and few of these patients were readmitted. This is the first longitudinal population-based analysis of SBO evaluating surgical versus nonsurgical management and outcomes, including mortality and readmissions.