A Novel Seimatosporium and Other Sporocadaceae Species Associated with Grapevine Trunk Diseases in Cyprus.

Besides well-known grapevine trunk disease (GTD)-related pathogens, there is an increased interest in wood-colonizing fungi that infect grapevines. During 2017-2018, a survey was conducted in Cyprus and wood samples were collected from vines exhibiting typical GTD symptoms. Based on morphological and multilocus phylogenetic analyses (ITS, LSU, bt2, tef1-a), four species in the Sporocadaceae family were described and typified; two in the genus of Seimatosporium: Seim. cyprium sp. nov. and Seim. vitis-viniferae and two in Sporocadus: Spo. kurdistanicus and Spo. rosigena. The teleomorph of Seim. cyprium sp. nov. was also described. Pathogenicity trials with representative isolates of each species were performed on woody stems of two-year-old potted grapevines for 12 months under field conditions. All isolates were pathogenic, causing dark brown to black vascular discoloration, extending upward and downward from the inoculation point. Sporocadus isolates were significantly more aggressive than Seimatosporium with lesion lengths ranging from 9.24 to 6.90 and 4.13 to 4.00 cm, respectively. Successful re-isolations were also evident for all species and isolates. Seim. cyprium sp. nov. is a newly described species, while Spo. kurdistanicus and Spo. rosigena are reported for the first time in Europe on Vitis vinifera, suggesting the potential role of Sporocadaceae in the GTDs complex.

Under Pressure: A Comparative Study of Botrytis cinerea Populations from Conventional and Organic Farms in Cyprus and Greece.

The highly heterogeneous nature of Botrytis cinerea provides adaptive benefits to variable environmental regimes. Disentangling pathogen population structure in anthropogenic agroecosystems is crucial to designing more effective management schemes. Herein, we studied how evolutionary forces exerted in different farming systems, in terms of agrochemicals-input, shape B. cinerea populations. In total, 360 B. cinerea isolates were collected from conventional and organic, strawberry and tomato farms in Cyprus and Greece. The occurrence and frequency of sensitivities to seven botryticides were estimated. Results highlighted widespread fungicide resistance in conventional farms since only 15.5% of the isolates were sensitive. A considerable frequency of fungicide-resistant isolates was also detected in the organic farms (14.9%). High resistance frequencies were observed for boscalid (67.7%), pyraclostrobin (67.3%), cyprodinil (65.9%), and thiophanate-methyl (61.4%) in conventional farms, while high levels of multiple fungicide resistance were also evident. Furthermore, B. cinerea isolates were genotyped using a set of seven microsatellite markers (simple sequence repeat [SSR] markers). Index of association analyses (Ia and rBarD) suggest asexual reproduction of the populations, even though the mating-type idiomorphs were equally distributed, indicating frequency-dependent selection. Fungicide resistance was correlated with farming systems across countries and crops, while SSRs were able to detect population structure associated with resistance to thiophanate-methyl, pyraclostrobin, boscalid, and cyprodinil. The expected heterozygosity in organic farms was significantly higher than in conventional, suggesting the absence of selective pressure that may change the allelic abundance in organic farms. However, genetic variance among strawberry and tomato populations was high, ranking host specificity higher than other selection forces studied.

Characterization of Phytophthora infestans Populations in Cyprus, the Southernmost Potato-Producing European Country.

Cyprus is the southernmost island country of Europe, located in the Mediterranean. Despite its limited area, potato production is considered an integral source of the national agricultural revenue. During 2010-2012, a late blight epidemic period for the country, the population structure of Phytophthora infestans was analyzed via a sample of 539 isolates collected from all of the main potato-cultivating regions of Cyprus. We determined mating type, mefenoxam sensitivity, and genetic polymorphism at 12 simple sequence repeat (SSRs) loci. Although both mating types were detected in the country, a gradual but dynamic shift toward A2 dominance was manifested over time. The pathogen population also demonstrated reduced sensitivity to the phenylamide fungicide, since 96.2% of the tested isolates had high (70.3%) and intermediate (25.9%) resistance to mefenoxam, which suggests that it should be replaced with other active ingredients in local disease management strategies. The genotypic analysis also revealed the predominance of the highly aggressive mefenoxam-insensitive EU_13_A2 lineage across the country, with a frequency of 79.2%. Other samples comprised an older lineage EU_2_A1 (19.5%), a very low proportion of EU_23_A1 (0.37%), and others that did not match any known lineage (0.92%). SSRs data supported triploid genomes among the dominant lineages, and patterns of their asexual population history were also apparent. A high subclonal variation of the 13_A2 population was detected, which suggested introduction events of this widespread genotype to Cyprus from major tuber-exporting countries. Present data indicate the severe impact of inoculum migration to the structure of the local population; thus, current phytosanitary procedures should be reconsidered and possibly attuned. This is the first comprehensive study to elucidate the diversity of P. infestans in Cyprus and could serve as a baseline for future monitoring of this highly adaptive plant pathogen, given that late blight management strategies should be constantly refined according to the traits of the dominant genotypes of P. infestans.

Antibody screening using a human iPSC-based blood-brain barrier model identifies antibodies that accumulate in the CNS.

Drug delivery across the blood-brain barrier (BBB) remains a significant obstacle for the development of neurological disease therapies. The low penetration of blood-borne therapeutics into the brain can oftentimes be attributed to the restrictive nature of the brain microvascular endothelial cells (BMECs) that comprise the BBB. One strategy beginning to be successfully leveraged is the use of endogenous receptor-mediated transcytosis (RMT) systems as a means to shuttle a targeted therapeutic into the brain. Limitations of known RMT targets and their cognate targeting reagents include brain specificity, brain uptake levels, and off-target effects, driving the search for new and potentially improved brain targeting reagent-RMT pairs. To this end, we deployed human-induced pluripotent stem cell (iPSC)-derived BMEC-like cells as a model BBB substrate on which to mine for new RMT-targeting antibody pairs. A nonimmune, human single-chain variable fragment (scFv) phage display library was screened for binding, internalization, and transcytosis across iPSC-derived BMECs. Lead candidates exhibited binding and internalization into BMECs as well as binding to both human and mouse BBB in brain tissue sections. Antibodies targeted the murine BBB after intravenous administration with one particular clone, 46.1-scFv, exhibiting a 26-fold increase in brain accumulation (8.1 nM). Moreover, clone 46.1-scFv was found to associate with postvascular, parenchymal cells, indicating its successful receptor-mediated transport across the BBB. Such a new BBB targeting ligand could enhance the transport of therapeutic molecules into the brain.

Impacts of the -1 Amino Acid on Yeast Production of Protein-Intein Fusions.

Expressing antibodies as fusions to the non-self-cleaving Mxe GyrA intein allows for site-specific chemical functionalization via expressed protein ligation. It is highly desirable to maximize the yield of functionalizable protein; and previously an evolved intein, 202-08, was identified that could increase protein fusion production in yeast. Given that the -1 amino acid residue upstream of inteins can affect cleavage efficiency, we examined the effects of amino acid variability at this position on 202-08 intein cleavage efficiency and secretion yield. Varying the -1 residue resulted in a wide range of cleavage behaviors with some amino acids yielding substantial autocleaved product that could not be functionalized. Autocleavage was noticeably higher with the 202-08 intein compared with the wild-type Mxe GyrA intein and resulted directly from the catalytic activity of the intein. Refeeding of production cultures with nitrogen base and casamino acids reduced, but did not eliminate autocleavage, while increasing protein-intein production up to seven-fold. Importantly, two amino acids, Gly and Ala, at the -1 position resulted in good cleavage efficiency with no undesirable autocleavage, and can be used in concert with refeeding strategies to increase total functionalizable protein yield for multiple protein fusion partners. Taken together, we describe an optimized yeast expression platform for protein-intein fusions. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2736, 2019.

Thermal Ablation in the Management of Adrenal Metastasis Originating from Non-small Cell Lung Cancer: A 5-year Single-center Experience.

BACKGROUND: Treatment of adrenal metastasis from lung carcinoma may prolong survival in the selected patients. However, not all patients can undergo surgery; thus, minimally invasive ablation procedures such as radiofrequency ablation (RFA) and microwave ablation (MWA) have gained acceptance as alternative treatment methods. This study summarized a 5-year single-center experience regarding the evaluation of safety and efficacy of computed tomography (CT)-guided thermal ablation in the management of adrenal metastasis originating from non-small cell lung cancer (NSCLC). METHODS: The data of NSCLC patients ablated for adrenal metastasis at the Department of Diagnostic Imaging and Interventional Radiology, General Hospital Sotiria, were retrospectively analyzed. Patients were divided into two groups: RFA group and MWA group according to the therapeutic approaches. Preprocedural blood tests included measurement of international normalized ratio, partial thromboplastin time, and platelet enumeration. A dual-phase contrast-enhanced spiral CT was performed immediately after the procedure to assess the immediate response after ablation and to screen for related complications. Follow-up was performed with CT or magnetic resonance imaging at 1, 3, 6 months and 1 year after ablation and every 6 months thereafter. RESULTS: A total of 99 ablation sessions in 71 patients with adrenal metastasis originating from NSCLC were included in the final analysis. Self-limited, postablation syndrome occurred in 16/99 (16.1%) of ablation sessions. All procedures were technically successful. Immediate postablation imaging showed no contrast enhancement of the ablated tumor in all patients. Follow-up imaging at 3 months revealed local tumor progression in 8 (22.8%) patients of the RFA group and 7 (19.4%) patients of MWA group, all of them underwent a second session successfully. The 1-year assessment revealed local recurrence of the ablated tumor in six patients (17.1%) of RFA group and seven patients (19.4%) of MWA group. Among these 71 patients, those with tumor size >3.5 cm had a higher local recurrence rate (65.2%, 15/23) than those with tumors ≤3.5 cm (16.7%, 8/48; P = 0.012). There was no significant difference in the median survival time between RFA (14.0 months) and MWA (14.6 months) groups (P > 0.05). CONCLUSIONS: RFA and MWA showed comparable efficacy and safety in adrenal metastasis treatment.

Protein engineering approaches for regulating blood-brain barrier transcytosis.

The blood brain barrier (BBB) presents a challenge for the delivery of brain therapeutics. Trans-BBB delivery methods that use targeting vectors to coopt the vesicle trafficking machinery of BBB endothelial cells have been developed, but these are often hampered by limited flux through the BBB. A solution to this problem lies in the semi-rational engineering of BBB targeting vectors. Leveraging knowledge of intracellular trafficking, researchers have begun to tune selected binding properties of the vector-receptor interaction. Engineered binding affinity, avidity and pH-sensitivity have been shown to affect binding, intracellular sorting and release, ultimately leading to increased brain uptake of the targeting vector and its associated cargo. However, each targeted receptor may exhibit differential responses to engineered binding properties, illustrating the need to better understand vector-receptor interactions and trafficking dynamics.

Thermal Ablation in the Management of Adrenal Metastasis Originating from Non-small Cell Lung Cancer: A 5-year Single-center Experience / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Treatment of adrenal metastasis from lung carcinoma may prolong survival in the selected patients. However, not all patients can undergo surgery; thus, minimally invasive ablation procedures such as radiofrequency ablation (RFA) and microwave ablation (MWA) have gained acceptance as alternative treatment methods. This study summarized a 5-year single-center experience regarding the evaluation of safety and efficacy of computed tomography (CT)-guided thermal ablation in the management of adrenal metastasis originating from non-small cell lung cancer (NSCLC).</p><p><b>METHODS</b>The data of NSCLC patients ablated for adrenal metastasis at the Department of Diagnostic Imaging and Interventional Radiology, General Hospital Sotiria, were retrospectively analyzed. Patients were divided into two groups: RFA group and MWA group according to the therapeutic approaches. Preprocedural blood tests included measurement of international normalized ratio, partial thromboplastin time, and platelet enumeration. A dual-phase contrast-enhanced spiral CT was performed immediately after the procedure to assess the immediate response after ablation and to screen for related complications. Follow-up was performed with CT or magnetic resonance imaging at 1, 3, 6 months and 1 year after ablation and every 6 months thereafter.</p><p><b>RESULTS</b>A total of 99 ablation sessions in 71 patients with adrenal metastasis originating from NSCLC were included in the final analysis. Self-limited, postablation syndrome occurred in 16/99 (16.1%) of ablation sessions. All procedures were technically successful. Immediate postablation imaging showed no contrast enhancement of the ablated tumor in all patients. Follow-up imaging at 3 months revealed local tumor progression in 8 (22.8%) patients of the RFA group and 7 (19.4%) patients of MWA group, all of them underwent a second session successfully. The 1-year assessment revealed local recurrence of the ablated tumor in six patients (17.1%) of RFA group and seven patients (19.4%) of MWA group. Among these 71 patients, those with tumor size >3.5 cm had a higher local recurrence rate (65.2%, 15/23) than those with tumors ≤3.5 cm (16.7%, 8/48; P = 0.012). There was no significant difference in the median survival time between RFA (14.0 months) and MWA (14.6 months) groups (P > 0.05).</p><p><b>CONCLUSIONS</b>RFA and MWA showed comparable efficacy and safety in adrenal metastasis treatment.</p>

Engineering an Anti-Transferrin Receptor ScFv for pH-Sensitive Binding Leads to Increased Intracellular Accumulation.

The equilibrium binding affinity of receptor-ligand or antibody-antigen pairs may be modulated by protonation of histidine side-chains, and such pH-dependent mechanisms play important roles in biological systems, affecting molecular uptake and trafficking. Here, we aimed to manipulate cellular transport of single-chain antibodies (scFvs) against the transferrin receptor (TfR) by engineering pH-dependent antigen binding. An anti-TfR scFv was subjected to histidine saturation mutagenesis of a single CDR. By employing yeast surface display with a pH-dependent screening pressure, scFvs having markedly increased dissociation from TfR at pH 5.5 were identified. The pH-sensitivity generally resulted from a central cluster of histidine residues in CDRH1. When soluble, pH-sensitive, scFv clone M16 was dosed onto live cells, the internalized fraction was 2.6-fold greater than scFvs that lacked pH-sensitive binding and the increase was dependent on endosomal acidification. Differences in the intracellular distribution of M16 were also observed consistent with an intracellular decoupling of the scFv M16-TfR complex. Engineered pH-sensitive TfR binding could prove important for increasing the effectiveness of TfR-targeted antibodies seeking to exploit endocytosis or transcytosis for drug delivery purposes.

Ectopic nephrogenic rest in the inguinal canal in a 6-month-old girl and a review of published studies.

Ectopic nephrogenic rests in the inguinal canal are rare. Usually discovered incidentally during surgery, these rests should raise the suspicion of an early extrarenal Wilms tumor. The differential diagnosis between the two entities is not only difficult but also essential, since they imply different treatment decisions. We report a rare case of an inguinal ectopic nephrogenic rest found in a 6-month-old girl and discuss the clinicopathological implications of this condition. The patient was admitted for a routine repair of a presumed inguinal hernia; during surgery, a nodular mass was noted in the inguinal canal. Pathological diagnosis confirmed the diagnosis of an extrarenal hyperplastic nephrogenic rest. Five previous cases of ectopic nephrogenic rests originating in the inguinal canal have been reported, all of which were associated with a patent processus vaginalis. In this case, the nephrogenic rest was not associated with a congenital inguinal hernia.

Intussusception in a premature neonate: a rare often misdiagnosed cause of intestinal obstruction.

Intestinal obstruction in neonatal period is an emergency caused by many surgical causes. An extremely rare surgical cause in this group of age is intussusception which can be easily confused with other surgical entities. In several reports, a significant number of the infants who were included in the study population were believed to have necrotizing enterocolitis (NEC). We present a rare cause of small intestine obstruction in a preterm female infant that can be easily misdiagnosed and confused preoperatively with other clinical entities particular for this period.