Nephrotoxic serum nephritis (NTN) models immune-mediated human glomerulonephritis and culminates in kidney inflammation and fibrosis, a process regulated by T lymphocytes. Tumor necrosis factor-α (TNF) is a key pro-inflammatory cytokine that contributes to diverse forms of renal injury. Therefore, we posited that TNF from T lymphocytes may contribute to NTN pathogenesis. Here, mice with T cell-specific deletion of TNF (TNF TKO) and wild-type (WT) controls were subjected to the NTN model. Compared to WTs, TNF TKO kidneys at day 14 of NTN have increased kidney injury and fibrosis. PD1⁺CD4⁺ T cell numbers and mRNA levels of IL-17A are elevated in NTN kidneys of TNF TKO mice, suggesting that augmented local Th17 responses in the TNF TKO kidney may exaggerate renal injury and fibrosis. In turn, we find increased accumulation of neutrophils in the TNF TKO kidneys during NTN. We conclude that TNF production in T lymphocytes mitigates NTN-induced kidney injury and fibrosis by inhibiting renal Th17 responses and infiltration of neutrophils.

The paper reports the purification and characterization of the first penicillin acylase from Bacillus subtilis. YxeI, the protein annotated as hypothetical, coded by the gene yxeI in the open reading frame between iol and hut operons in B. subtilis was cloned and expressed in Eshcherichia coli, purified and characterized. The purified protein showed measurable penicillin acylase activity with penicillin V. The enzyme was a homotetramer of 148 kDa. The apparent K(m) of the enzyme for penicillin V and the synthetic substrate 2-nitro-5-(phenoxyacetamido)-benzoic acid was 40 mM and 0.63 mM, respectively, and the association constants were 8.93×10(2) M(-1) and 2.51×10(5) M(-1), respectively. It was inhibited by cephalosporins and conjugated bile salts, substrates of the closely related bile acid hydrolases. It had good sequence homology with other penicillin V acylases and conjugated bile acid hydrolases, members of the Ntn hydrolase family. The N-terminal nucleophile was a cysteine which is revealed by a simple removal of N-formyl-methionine. The activity of the protein was affected by high temperature, acidic pH and the presence of the denaturant guanidine hydrochloride.

Objective: The purpose of this study was to explore the prevalence of thyroid nodules (TN) and metabolic syndrome (MS) and to analyze the correlation between TN and the components of MS.

Methods: A total of 1526 subjects were divided into two groups: a TN group and a non-thyroid nodules (NTN) group. The height, weight, blood pressure, fasting blood glucose level, fasting plasma insulin level, serum lipid profile, uric acid level, serum thyroid-stimulating hormone (TSH) level, free triiodothyronine (FT3) level, and free thyroxine (FT4) level of each patient were measured. Insulin resistance (IR) was estimated by homeostasis model assessment of insulin resistance (HOMA-IR). Fatty liver and TN were detected by color Doppler ultrasonography.

Results: (i) The overall prevalence of TN was 39.5%; it was significantly higher in women than in men (P < 0.01) and progressively increased with age in both sexes. (ii) The overall prevalence of MS was 25.6%; it was significantly higher in men than in women (P < 0.01) and progressively increased with age in both sexes. (iii) FT3 was significantly lower in the TN group than in the NTN group (P < 0.01). (iv) BMI, triglycerides, and HOMA-IR were higher in the TN group than in the NTN group (P < 0.05). (v) The existence of TN was significantly associated with overweight/obesity (OR = 1.03, 95% CI = 1.024-1.089), and with insulin resistance (IR) (OR = 1.98, 95% CI = 1.645-2.368), after adjusting for age and sex.

Conclusions: The prevalence of thyroid nodules and metabolic syndrome in the Nanchang area increases with age, and overweight/obesity and IR in patients are associated with thyroid nodules.

Keywords: correlation; insulin resistance; metabolic syndrome; obesity; thyroid nodules.

Lipid peroxidation (LPO) stimulated by ascorbate was studied in renal cortex of 20 rats with nephrotoxic nephritis (NTN) and of 9 rats with proteinuria induced by a 3-day course of i. p. injections of the human serum albumin. At the early stages of NTN (0.5 h. and 3 h.) LPO activities were of the same values as in control rats. A small decrease in renal cortex LPO was found on the 4-th day of NTN when nephrotic syndrome has been developed. A significant reduction in LPO activity was observed on the 16-th day of NTN characterized by a more pronounced nephrotic syndrome. LPO activity in renal cortex of the rats with albumin overload proteinuria was also reduced. An inhibitory effect of proteinuria on LPO activity in kidney is discussed.

Non-selective inhibitors of spleen tyrosine kinase (SYK) efficiently suppress disease in T cell-dependent models of crescentic glomerulonephritis. However, the therapeutic potential of selective SYK inhibitors in this disease has not been established. In addition, we lack knowledge regarding SYK expression in non-myeloid cells in glomerulonephritis. We addressed these two issues in a rat model of nephrotoxic serum nephritis (NTN) using a SYK inhibitor, GS-492429. Disease was induced in Sprague-Dawley rats (Study 1) or Wistar-Kyoto (WKY) rats (Study 2) by immunization with sheep IgG and administration of sheep anti-rat nephrotoxic serum. Animals were untreated or received GS-492429 (30 mg/kg/bid) or vehicle treatment from 2 h before nephrotoxic serum injection until being killed 3 or 24 h later (Study 1) or 14 days later (Study 2). Two-colour confocal microscopy found that SYK expression in NTN kidney was restricted to myeloid cells and platelets, with no evidence of SYK expression by T cells, mesangial cells, podocytes or tubular epithelial cells. In Study 1, GS-492429 treatment significantly reduced glomerular neutrophil and macrophage infiltration, with protection from glomerular thrombosis and proteinuria. In Study 2, GS-492429 treatment reduced glomerular crescent formation by 70% on day 14 NTN in conjunction with reduced glomerular thrombosis, glomerulosclerosis and tubular damage. This was accompanied by a marked reduction in markers of inflammation (CCL2, TNF-α, NOS2, MMP-12). Importantly, the protective effects of GS-492429 were independent of T cell infiltration and activation and independent of JAK/STAT3 signalling. In conclusion, this study demonstrates that a SYK inhibitor can suppress the development of crescentic glomerulonephritis through effects upon myeloid cells and platelets.

In 1999, clary sage plants (Salvia sclarea L.) at the Herb Garden of Casola Valsenio (Emilia-Romagna Region, northern Italy) exhibited malformed leaves with yellow spots and line patterns. Sap from leaves of symptomatic sage plants caused symptoms in inoculated Chenopodium amaranticolor Coste et Reyn. plants (local chloro-necrotic lesions developed 7 to 10 days after inoculation) and Nicotiana tabacum L. 'White Burley' and 'Samsun' plants (systemic veinal necrosis developed ≈ 2 weeks after inoculation). Leaves from symptomatic sage plants tested positive for Potato virus Y (PVY) based on immunoelectron microscopy, gold-labeled decoration, and protein A sandwich enzyme-linked immunosorbent assay (ELISA), using antiserum to PVY (PVAS 50a, American Type Culture Collection, Manassas, VA). Double-antibody sandwich-ELISA, using specific monoclonal antibodies (BioReba AG, Reinach, Switzerland) to the tobacco veinal necrosis strain group of PVY (PVY-N), revealed that the PVY isolate from sage belonged to this group. Immunocapture-reverse transcription-polymerase chain reaction, using specific primers for PVY and the tuber necrotic strain of PVY (PVY-NTN), further classified the sage isolate as PVY-NTN (1). PVY-NTN causes serious damage to potato in Europe. Clary sage, one of the most important aromatic plants cultivated worldwide as a source of essential oils, represents a new natural host of PVY-NTN. Reference: (1) H. L. Weidemann and E. Maiss. J. Plant Dis. Prot. 103:337, 1996.

During the last 2 years, potato (Solanum tuberosum L.) tubers displaying superficial necrotic arcs and rings were found in central Portugal. These symptoms increased during storage, and diminished tuber quality of ware (fresh-market) potatoes; however, no internal necrosis, which is typical for infections caused by tobacco rattle virus or potato mop top virus, was observed. The symptoms led to the preliminary diagnosis of potato tuber ringspot disease (PTNRD), caused by a tuber necrosis (TN)-inducing isolate of the tobacco veinal necrosis strain group of potato virus Y (PVY^N) that was named PVY^NTN. The occurrence of PVY^NTN has been reported by a number of European countries. Suspect PTNRD tubers of the cv. Monalisa were obtained from several Portuguese potato growers and were tested with polyclonal antibodies (pabs) that reacted generally with PVY, and with monoclonal antibodies (mabs) raised against PVY^N. Serogical tests were carried out in a double antibody sandwich (DAS) enzyme-linked immunosorbent assay (ELISA) with pabs and in a triple antibody sandwich (TAS) ELISA when mabs were used. As a result, the tubers were found to be infected with a virus isolate belonging to the PVY^N strain group. Since PVY^NTN cannot be distinguished serologically from other members of the PVY^N strain group due to the similarities of their coat proteins (1), reverse transcription-polymerase chain reaction combined with immunocapture was applied for diagnostic purposes. The olignucleotide primers used were located in the 5' non-coding region at nucleotide 103 and in the adjacent P1 protein gene coding region at position 919. This primer pair can be used to distinguish PVY^NTN from other members of the PVY^N strain group (2). Tests were carried out with plant sap from tubers and from plants grown from eye-cuttings and also from tobacco plants that were inoculated with plant sap of these potato tubers and plants. Control samples included sap from un-infected tobacco plants and from tobacco plants infected with a PVY^N isolate and with the PVY^NTN type strain "Hungary". The expected amplification product of 835 bp appeared in the agarose gel with samples originally obtained from the tubers and with the PVY^NTN control but not with the PVY^NTN control, indicating that the tuber symptoms in potato cv. Monalisa were caused by infections with PVY^NTN. This is the first report of the occurrence of PVY^NTN in Portugal. References: (1) T. Dalmay and E. Balazs. Nucleic Acids Res. 18: 6721, 1990. (2) H. L. Weidemann and E. Maiss. Z. Pflanzenkr. Pflanzenschutz 103:337, 1996.

BACKGROUND

Virus-induced gene silencing (VIGS) is an optimal tool for functional analysis of genes in plants, as the viral vector spreads throughout the plant and causes reduced expression of selected gene over the whole plant. Potato (Solanum tuberosum) is one of the most important food crops, therefore studies performing functional analysis of its genes are very important. However, the majority of potato cultivars used in laboratory experimental setups are not well amenable to available VIGS systems, thus other model plants from Solanaceae family are used (usually Nicotiana benthamiana). Wild potato relatives can be a better choice for potato model, but their potential in this field was yet not fully explored. This manuscript presents the set-up of VIGS, based on Tobacco rattle virus (TRV) in wild potato relatives for functional studies in potato-virus interactions.

RESULTS

Five different potato cultivars, usually used in our lab, did not respond to silencing of phytoene desaturase (PDS) gene with TRV-based vector. Thus screening of a large set of wild potato relatives (different Solanum species and their clones) for their susceptibility to VIGS was performed by silencing PDS gene. We identified several responsive species and further tested susceptibility of these genotypes to potato virus Y (PVY) strain NTN and N. In some species we observed that the presence of empty TRV vector restricted the movement of PVY. Fluorescently tagged PVY(N)-GFP spread systemically in only five of tested wild potato relatives. Based on the results, Solanum venturii (VNT366-2) was selected as the most suitable system for functional analysis of genes involved in potato-PVY interaction. The system was tested by silencing two different plant immune signalling-related kinases, StWIPK and StMKK6. Silencing of StMKK6 enabled faster spreading of the virus throughout the plant, while silencing of WIPK had no effect on spreading of the virus.

CONCLUSIONS

The system employing S. venturii (VNT366-2) and PVY(N)-GFP is a suitable method for fast and simple functional analysis of genes involved in potato-PVY interactions. Additionally, a set of identified VIGS responsive species of wild potato relatives could serve as a tool for general studies of potato gene function.

More than 50 isolates of Potato virus Y (PVY) with characteristics of strains that cause tobacco veinal necrosis (PVY^N) were obtained from potatoes (Solanum tuberosum L.) grown in the northwestern United States. These isolates are being characterized at the biological and molecular levels. Isolate RR1 was obtained from leaves of potato cv. Ranger Russet showing distinct mottling and leaf deformity, which is in contrast to the leaf-drop and necrosis usually observed with ordinary strains of PVY (PVY^O) in this variety. Isolate AL1 was obtained from tubers of potato cv. Alturas showing distinct internal light brown rings and blotches. When RR1 and AL1 were transmitted to tobacco (Nicotiana tabacum L. cvs. Samsun NN and 423), they caused systemic veinal necrosis, including stem and petiole lesions typical of PVY^N strains (2). Symptoms induced by RR1 and AL1 on tobacco appeared 9 to 11 days after inoculation, whereas some other isolates caused delayed veinal necrosis. All isolates that produced veinal necrosis on tobacco were detectable with PVY polyclonal antisera. Potato virus X was not detected by enzyme-linked immunosorbent assay in tobacco plants showing veinal necrosis. Some isolates, including AL1, failed to react in serological tests using PVY^N-specific monoclonal antibodies obtained from three commercial sources. Other isolates, including RR1, were detectable with these monoclonal antibodies. Reverse transcription-polymerase chain reaction (RT-PCR) products obtained with primers specific for the coat protein (CP) open reading frame (ORF) were cloned and sequenced. AL1 possesses a CP more closely related to PVY^O type isolates, which would account for its failure to react with PVY^N monoclonal antibodies. In this regard, AL1 is similar to the PVY^N-Wilga isolate (1). Other isolates that are detectable with the PVY^N monoclonal antibodies possess a CP more consistent with N strains of the virus. Results of RT-PCR tests using primers derived from the P1 ORF sequence (3), and the restriction enzyme analysis and sequencing of the RT-PCR products, all suggest that AL1 and RR1 are related to European-type members of PVY tuber necrotic (NTN) or N strains. However, other isolates under investigation appear to be more closely related to previously reported North American NTN types (3). The symptomatology of these viruses on tobacco and potato, and the serological and molecular data clearly show that at least two distinct variants of PVY^N have been found for the first time in a major potato production area of the United States, and pose a potential threat to the potato industry. References: (1) B. Blanco-Urgoiti et al. Eur. J. Plant Pathol. 104:811, 1998. (2) J. A. de Bokx and H. Huttinga. Potato virus Y. Descriptions of Plant Viruses. No. 242, CMI/AAB, Surrey, England, 1981. (3) R. P. Singh et al. Can J. Plant Pathol. 20:227, 1998.

The objective of this study was to examine the reaction of 12 Capsicum breeding lines to NTN strain of Potato virus Y (PVY(NTN)) and 16 lines to Obuda pepper virus (ObPV). Inoculated plants were symptomatologically and serologically checked for virus infection. Back inoculation was also carried out to Nicotiana tabacum 'Xanthi-nc' and N. tabacum 'Samsun' as indicator plants. Out of the 12 lines tested four (32.Bogyiszlói, 4/99 F2, 17/99 F2 and VI-61 in.) proved to be resistant (immune) to PVY(NTN). Seven Capsicum lines (9/99 F2, 17/99 F2, V-21 = 28/98 F3, V-28 = 36/98 F3, V-3 = 7/98 F2, V-6 = 13/98 F2, and V-10 = 17/98 F2) showed hypersensitive reaction to ObPV. Other breeding lines were susceptible to ObPV infection. One line (17/99 F2) showed immunity to PVY(NTN) and hypersensitivity to ObPV at the same time, therefore this one is considerably valuable for breeding pepper varieties for multivirus resistance.

Potato (Solanum tuberosum L.) is widely grown as a staple food and cash crop in Tajikistan and is an important food security crop in the country. In June 2011, we conducted a survey of potatoes in farmers' fields in the Buston and Dushanbe regions (about 200 miles apart) of Tajikistan. Potato plants with stunted growth and leaves showing chlorotic spots, curling, and necrotic spots and rings were observed with the disease incidence monitored in 10 fields each in Buston and Dushanbe areas varying between 10 and 60%. Representative samples from symptomatic plants tested positive for Potato virus Y (PVY) using virus-specific immunostrips (Agdia Inc., Elkhart, IN). Leaf samples from symptomatic plants were collected from Buston and Dushanbe areas, imprinted on FTA Classic Cards (Whatman International Ltd., Maidstone, UK), air dried, and shipped to the lab at Washington State University for confirmatory diagnostic tests. Total nucleic acids were eluted from FTA cards (1) and subjected to reverse transcription (RT)-PCR with primers (PVY/Y4A and PVY/Y3S) specific to the coat protein of PVY (3). Samples infected with PVY ordinary strain (PVY^O), tuber necrosis strain (PVY^NTN), tobacco veinal necrosis strains (PVY^EU-N and PVY^NA-N), and a recombinant strain (PVY^N:O) were included as references to validate RT-PCR results. A single DNA product of approximately 480 bp was amplified from potato samples that tested positive with PVY-specific immunostrips. The amplified fragments from two samples from Dushanbe and six from Buston areas were cloned separately into pCR2.1 (Invitrogen Corp., Carlsbad, CA) and two independent clones per amplicon were sequenced from both orientations. Pairwise comparison of these sequences showed 90 to 100% identity among the cloned amplicons (GenBank Accession Nos. JQ743609 to JQ743616) and 90 to 100% with corresponding nucleotide sequence of reference PVY strains (GenBank Accession Nos. JQ743617 to JQ743621). A global phylogenetic analysis of sequences revealed the presence of PVY^O in both samples from Dushanbe and one sample from Buston regions and presence of PVY^NTN in the remaining five samples from the Buston region. Because of the possible occurrence of mixed infections of PVY strains (2), further studies are needed to determine the presence of mixed infections of two or more strains of PVY and their specificity to potato cultivars. To our knowledge, this study represents the first confirmed report of two distinct strains of PVY in potato in Tajikistan. The occurrence of PVY^NTN, a quarantine pathogen in many countries (2), warrants additional investigations to improve sanitary status of potato fields and to facilitate the availability of virus-free seed in clean plant programs for significant yield increases in Tajikistan. References: (1) O. J. Alabi et al. J. Virol. Methods 154:111, 2008. (2) S. Gray et al. Plant Dis. 94:1384, 2010. (3) R. P. Singh et al. J. Virol. Methods 59:189, 1996.

There is a growing demand in both food quality and quantity, but as of now, one-third of all food produced for human consumption is lost due to pests and other pathogens accounting for roughly 40% of pre-harvest loss in potatoes. Pathogens in potato plants, like the Erwinia bacteria and the PVY^NTN virus for example, exhibit symptoms of varying severity that are not easily captured by pixel-based classes (as these ignore shape, texture, and context in general). The aim of this research is to develop an object-based image analysis (OBIA) method for trait retrieval of individual potato plants that maximizes information output from Unmanned Aerial Vehicle (UAV) RGB very high resolution (VHR) imagery and its derivatives, to be used for disease detection of the Solanum tuberosum. The approach proposed can be split in two steps: (1) object-based mapping of potato plants using an optimized implementation of large scale mean-shift segmentation (LSMSS), and (2) classification of disease using a random forest (RF) model for a set of morphological traits computed from their associative objects. The approach was proven viable as the associative RF model detected presence of Erwinia and PVY pathogens with a maximum F1 score of 0.75 and an average Matthews Correlation Coefficient (MCC) score of 0.47. It also shows that low-altitude imagery acquired with a commercial UAV is a viable off-the-shelf tool for precision farming, and potato pathogen detection.

A virus was isolated from joyweed (Alternanthera tenella Colla-Amaranthaceae), a common weed in tropical and sub-tropical regions. Examination by electron microscopy showed long flexuous particles with an average length of 756 nm in crude sap. Serological results showed positive reaction with antisera to PVY-O. A fragment of 1772 nucleotides was sequenced. The CP sequence shares 76% of identity with the CP of Potato virus Y strain NTN. These results confirm that the virus is a new potyvirus infecting A. tenella, and the name Alternanthera mild mosaic virus (AltMMV) is proposed.

Potato (Solanum tuberosum) is an important vegetable crop in Indonesia. A small survey was conducted for virus diseases in November 2011 in Lembang, West Java, as part of assessing the sanitary status of potatoes produced in farmers' fields. Among the six potato fields surveyed, one field had nearly 20% of plants displaying stunted growth with leaves showing mild chlorotic spots and reduced size of lamina. Tubers harvested from symptomatic plants showed no necrosis symptoms. Symptomatic leaves from three representative potato plants were positive for Potato virus Y (PVY) when tested with PVY-specific immunostrips (Agdia Inc., Elkhart, IN). Leaf samples from virus-positive plants were imprinted on FTA Classic Cards (Whatman International Ltd., Maidstone, UK), air dried, and shipped to Washington State University for confirmatory diagnostic tests. Total nucleic acids were eluted from FTA cards (1) and subjected to reverse transcription (RT)-PCR using primers (PVY/Y4A and PVY/Y3S) specific to the coat protein (CP) of PVY (3). Nucleic acid extracts from samples infected with PVY ordinary strain (PVY^O), tuber necrosis strain (PVY^NTN), tobacco veinal necrosis strains (PVY^EU-N and PVY^NA-N), and a recombinant strain (PVY^N:O) were included as standards to validate RT-PCR assays. The approximately 480-bp DNA fragment, representing a portion of the CP, amplified in RT-PCR was cloned into pCR2.1 (Invitrogen Corp., Carlsbad, CA). DNA isolated from four independent recombinant clones was sequenced from both orientations. Pairwise comparison of these sequences (GenBank Accession Nos. KF261310 to 13) showed 100% identity among themselves and 93 to 100% identity with corresponding sequences of reference strains of PVY available in GenBank (JQ743609 to 21). To our knowledge, this study represents the first confirmed report of PVY in potato in West Java, Indonesia. Studies are in progress to assess the prevalence of PVY in other potato-growing regions of Indonesia and document the presence of different strains of the virus (2). Since the majority of farmers in Indonesia plant seed selected from their previous potato crop, there is an increased risk of primary and secondary spread of PVY through the informal seed supply system, leading to its increased significance to potato production in Indonesia. Therefore, strengthening foundation seed potato and supply chain programs will promote the production of virus-free potatoes in Indonesia. References: (1) O. J. Alabi et al. Plant Dis. 96:107, 2012. (2) A. Karasev and S. M. Gray. Am. J. Potato Res. 90:7, 2013. (3) R. P. Singh et al. J. Virol. Methods 59:189, 1996.

Potato (Solanum tuberosum L.) is an important vegetable crop in Jordan, occupying second position after olives. In 2012, potatoes were planted on about 6,000 ha with a production of about 141,000 t (2). Potato virus Y (PVY) is a serious problem for potato production worldwide. Recombinant strains of the virus were reported to cause tuber necrotic ringspot disease (PTNRD) in many potato-growing regions of the world. In the last few years, a new recombinant PVY^NTN-NW that belongs to PVY^Z (3) has been reported in the neighboring Syria. It included three recombination patterns, SYR-I, SYR-II, and SYR-III, and caused severe PTNRD (1). Since PVY is easily transmitted from one region to another by aphid vectors and infected potato seeds, this study was initiated to investigate the possible occurrence of PVY strains in Jordan. In October 2013, 33 leaf samples were collected from symptomatic potato plants cv. Spunta from Wadi Rum, Jordan (GPS coordinates 29°31'37.76″ N, 35°42'48.75″ E), the largest potato-producing area in Jordan. Sampled plants displayed leaf mottling and yellowing, symptoms similar to those caused by PVY. All samples were tested for PVY by DAS-ELISA using the ELISA kit (monoclonal cocktail) developed by BIOREBA (Reinach, Switzerland) to detect all PVY isolates. Twenty-nine samples were found positive for PVY by ELISA. To confirm virus infection, total RNA was extracted from all ELISA-positive samples and used as template in uniplex RT-PCR using strain-specific primers (1). The band pattern of PCR amplicons showed that 12 samples were infected with PVY^NTN-NW genotype SYR-III and produced bands of 1,085, 441, and 278 bp. One sample was infected with PVY^NTN (A) and produced bands of 1,307, 633, and 441 bp, and one other sample was infected with PVY^NTN-NW genotype SYR-II and produced bands of 1,085 and 441 bp. Mixed infection with PVY^NTN-NW genotype SYR-III and PVY^NTN (B) was also detected in one sample producing bands of 278, 441, 1,085, and 1,307 bp. To confirm infection with the recombinant strains, PCR fragments of 278 bp amplified from three samples and 1,085 bp obtained from another three samples were directly sequenced and sequences were deposited in GenBank under accession numbers KJ159968, KJ159969, and KJ159970 for the 278-bp fragment and KJ159974, KJ159975, and KJ159976 for the 1,085-bp fragment. Sequence comparison with other PVY strains available in the NCBI database showed that the 278-bp fragment had the highest nucleotide sequence identity (100%) with PVY isolates SYR-III-A26 (AB461467) and SYR-III-2-4 (AB461457) from Syria. BLAST searches also showed that the 1,085-bp fragment shared 99% nucleotide identities with PVY isolates SYR-II-L3 (AB461482) and SYR-II-Be4 (AB461474) from Aleppo, Syria. To our knowledge, this is the first report of PVY recombinants in Jordan, and the first report of PVY^NTN-NW recombinants infecting potato crop outside Syria. Since Europe is the main supplier of potato seeds for farmers in Jordan and Syria, the introduction of PVY^NTN-NW to the region could have happened through infected potato seeds. Results of this study create new challenges for potato growers in Jordan as well as other countries in the region. References: (1) M. Chikh Ali et al. J. Virol. Methods 165:15, 2010. (2) FAO. http://faostat.fao.org/ (3) A. V. Karasev and S. M. Gray. Ann. Rev. Phytopathol. 51:571, 2013.

In 2005, weekly rain samples collected at 124 National Atmospheric Deposition Program/National Trends Network (NADP/NTN) sites in the eastern and central United States were screened for Asian soybean rust (ASR; Phakopsora pachyrhizi) urediniospores. Application of a quantitative polymerase chain reaction method detected P. pachyrhizi DNA in the filter residue of rain samples collected during the week of 19 to 26 July 2005 in Minnesota, Missouri, and South Dakota. To determine the geographic origin of ASR urediniospores in those weekly composite samples, back air trajectories of the lifted condensation and mixed boundary layers were calculated for each rain event within the week, by sampling site. The calculations, based on the hybrid single-particle lagrangian integrated trajectory model, pointed to source areas in eastern and southern Texas. In a separate case, DNA of P. pachyrhizi was detected in a 28 June to 5 July 2005 rain sample from an eastern Texas site. Back trajectories pointed to southern Texas and the Yucatan Peninsula in Mexico as potential source areas of ASR urediniospores. Vertical motions of those back trajectories indicated a ventilation of the boundary layer in the upwind areas, suggesting the possible injection of urediniospores into the free troposphere where they can be transported for long distances before wet deposition.

Acid-labile subunit (ALS) is a component of the 150-kDa insulin-like growth factor-binding protein-3 (IGFBP-3) complex, which, by sequestering the majority of IGFs-I and -II and thereby prolonging the half-life of them in plasma, serves as a circulating reservoir of IGFs in mammalian species. A pGEX-2T plasmid and a baculovirus expression constructs harboring a coding sequence for glutathione-S-transferase (GST)-porcine ALS (pALS) fusion protein were expressed in BL21(DE3) E. coli and Sf9 insect cells, respectively. The expressed protein was purified by glutathione or Ni-NTN affinity chromatography, followed by cleavage of the fusion protein using Factor Xa. In addition, pALS and hIGFBP-3 were also produced in small amounts in the Xenopus oocyte expression system which does not require any purification procedure. A 65-kDa pALS polypeptide was obtained following the prokaryotic expression and the enzymatic digestion, but biochemical characterization of this polypeptide was precluded because of an extremely low expression efficiency. The baculovirus as well as Xenopus-expressed pALS exhibited the expected molecular mass of 85 kDa which was reduced into 75 and 65 kDa following deglycosylation of Asn-linked carbohydrates by Endo-F glycosidase, indicating that the expressed pALS was properly glycosylated. Moreover, irrespective of the source of pALS, the recombinant pALS and hIGFBP-3 formed a 130-kDa binary complex which could be immunoprecipitated by anti-hIGFBP-3 antibodies. Collectively, results indicate that an authentic pALS protein can be produced by the current expression systems.

This study was conducted to determine the effects of dietary addition of tea saponins (TS) and soybean oil (SO) on fatty acid profile and gene expression of stearoyl-CoA desaturase (SCD) in longissimus dorsi (LD) muscle of growing lambs. Thirty-two Huzhou lambs were assigned to four dietary treatments in a 2×2 factorial arrangement with main effects of TS (0 or 3 g/d) and SO (0 or 30 g/kg of diet DM). The diet without additives was considered as NTNS (no TS or SO). After a feeding trial for 60 d, four lambs of each treatment were slaughtered to collect the samples of LD muscle. Percentage of trans-11 vaccenic acid was enhanced (p<0.05) in muscle of lambs fed TS and SO. The proportion of total conjugated linoleic acid (CLA) was increased (p<0.05) by SO, but decreased (p<0.05) by TS in LD muscle. The percentage of total saturated fatty acids in muscle was decreased (p<0.05) by addition of TS and SO, while addition of SO increased (p<0.05) the percentage of total polyunsaturated fatty acids. The ratio of cis-9, trans-11 CLA to tran-11 vaccenic acid was decreased (p<0.05) by TS, but increased (p<0.05) by SO. The same effects were observed in SCD mRNA expression. From these results it is indicated that including TS and SO in the diet of growing lambs affect the fatty acid profiles of LD muscle and that the proportion of cis-9, trans-11 CLA in the muscle influenced by TS and SO may be related to the SCD gene expression.

The contents and forms of nitrogen (N), phosphorus (P), and organic carbon (C) were determined with 40 cm (approximately 1600 s) core sediments from Erhai Lake on the Yungui Plateau of China as the sample. The vertical distribution characteristics, coupling relationships and ecological indicator significance of C-N-P were studied and identified. The results showed that the total organic carbon (TOC), total nitrogen (TN), and total phosphorus (TP) contents were in the ranges of 1436-8255 mg·kg^-1, 1287-5462 mg·kg^-1, and 870.26-1507.74 mg·kg^-1, respectively. In the Erhai Lake sediments, the main forms of TOC, organic nitrogen (ON) and organic phosphorus (OP) were humus, TN, and TP, respectively. The deposition of C, N, and P in the Erhai Lake sediments was divided into four periods. In the initial development period (from 40 to 23 cm), C, N and P were deposited and released synchronously; the main form of N was nontransferable total nitrogen (NTN), and that of P was inorganic phosphorus (IP). In the ecological recovery period (from 22 to 14 cm), C and N were deposited synchronously, and their deposition amounts were more than that of P. C, N and P were released synchronously. The main form of N was transferable total nitrogen (TTN), and that of P was IP. In the rapid economic growth period (from 13 to 0 cm), C, N, and P were deposited and released synchronously; the main form of N was NTN, and that of P was OP. In comparison to the other periods, this period was a period of higher active soil organic carbon (ASOC). In the integrated management period (surface sediment), C and N were deposited and released synchronously, and their deposition amounts were greater than that of P. The main form of N was NTN, that of P was OP, and the ASOC content was high. When exogenous inputs were the main sources of C, N, and P, the deposition forms of P and N were mainly OP and NTN, respectively, and those of IP and TTN were calcium-bound P (Ca-P) and ion-exchange form N (IEF-N), respectively. When endogenous inputs were the main sources of C, N and P, the deposition forms of P and N were mainly IP and TTN, respectively, and those of IP and TTN were Fe/Al-P and weak acid extractable form (WAEF-N), respectively. The content ratio of Ca-P and Fe/Al-P, as well as that of IEF-N and WAEF-N, could reflect the changes in the contribution of endogenous and exogenous sources to the Erhai Lake sediments.

Neoadjuvant chemotherapy (NC) facilitates breast conservation in women with large tumors, which are common in our inner city breast clinic. We performed this review of our NC breast cancer experience, which includes a disproportionate number of triple negative patients. Patients treated with NC were divided into two groups based on their tumor's receptor status. Patients with tumors negative for estrogen, progesterone, and HER2-neu were considered triple negative (TN) and patients with positive staining for any of these receptors were considered nontriple negative (NTN). Response to NC was considered a complete response (CR) if no residual tumor was detected at surgery, partial response (PR) if the height and width was reduced by at least 50 per cent, and no response (NR) for anything less than a PR. Differences were assessed by χ(2) analysis and Student's t test. We identified 30 patients treated with NC (11 TN and 19 NTN). Twenty-one patients (70%) were African American (11/11 TN and 10/19 NTN; P = 0.01). Age (46.8 ± 6.0 years TN vs 49.5 ± 11.7 years NTN), response rates (18% NR, 55% PR, and 27% CR TN; 37% NR, 42% PR, and 21% CR NTN), and node positivity (64% TN vs 74% NTN) were statistically similar. Two TN (20%) and seven (41%) NTN patients underwent breast conservation therapy. Our results demonstrate the association of African American race and TN breast cancer. TN cancers respond similarly to NC when compared with NTN, allowing for tumor downstaging and possible breast conservation surgery.

In the process of developing a pH-stable, highly crosslinked stationary phase using the thiol-yne reaction, a new charge transfer stationary phase was discovered. The first step in the preparation of the crosslinked phase is to attach 1,4-diethynylbenzene (DEB) to thiol functionalized silica particles using the thiol-yne reaction. Upon preparation of that phase, we noticed that the color of the particles was different when the modified particles were wet with aromatic solvents in comparison to wetting with nonaromatic or aqueous solvents. This color change was still apparent upon crosslinking the DEB with 1,6-hexanedithiol to create the crosslinked stationary phase. The chromatographic selectivity for the flat triphenylene over the bulkier o-terphenyl, α_T/OT is an indicator of shape selectivity. The crosslinked phase⬢s α_T/OT is 4.91 ± 0.08, almost twice that of the most shape-selective reversed phase column. The difference of the entropy contributions to retention free energy between the two compounds, ΝTΝS° at 298⬰K, is statistically indistinguishable from zero, (↙0.1⬰±⬰0.9⬰kJ/mol) leading us to believe that the observed shape selectivity is not consistent with the slot model. To test the hypothesis that the DEB-thiol adduct, a 4-ethynyl styryl sulfide (ESS), was responsible for the observed behavior, we prepared a low coverage ESS-containing phase which, unlike higher density, crosslinked, or polymeric phases, should not display shape selectivity based on ⬓slots⬽. With the ESS phase the shape selectivity remained high, with α_T/OT⬰=⬰3.23⬰±⬰0.01. The ESS ligand has electron donating characteristics based on the selectivity for nitrobenzene compared to benzene: 1.83⬰±⬰0.10 on the ESS phase vs 0.64 ± 0.01 on a commercial C18 stationary phase. This shows that the thiol-yne based ESS stationary phase has electron donating charge transfer characteristics.

Background: Certification of seed potato as free of viruses is essential for stable potato production. Among more than 30 virus species infecting potato, potato leafroll virus (PLRV), potato virus S (PVS), potato virus X (PVX), and potato virus Y (PVY) predominate worldwide and should be the targets of a high-throughput detection protocol for seed potato quarantine.

Results: We developed an assay based on one-step real-time multiplex reverse transcription-polymerase chain reaction (mRT-PCR) with melt curve analysis for the four viruses and one internal control, potato elongation factor 1 alpha gene (EF1α). Virus-specific primers were derived from conserved regions among randomly selected representatives considering viral genomic diversity. Our assay simultaneously detected representative Japanese isolates of PLRV, O lineage of PVS, PVX, and NTN strain of PVY. The variability of melting temperature (Tm) values for each virus was confirmed using Japanese isolates, and virus species could be identified by the values of 87.6 for PLRV, 85.9 for PVX, 82.2 (Ordinary lineage) to 83.1 (Andean lineage) for PVS, and 79.4 (NA-N strain) to 80.5 (O strain and NTN strain) for PVY on average. The reliability of calculation was validated by comparing the calculated Tm values and measured Tm values and the values had a strong linear correlation (correlation of determination: R² = 0.9875). Based on the calculated Tm values, representative non-Japanese isolates could also be identified by our assay. For removing false positives, two criteria were set for the evaluation of result; successful amplification was considered as 30.0 ≥ threshold cycle value, and the virus-specific peak higher than the EF1α-specific peak was considered as positive. According to these criteria, our assay could detect PLRV and PVS from 100-fold dilution of potato leaf homogenate and PVX and PVY from 1000-fold in a model assay.

Conclusion: This new high-throughput detection protocol using one-step real-time mRT-PCR was sensitive enough to detect viruses in a 100-fold dilution of singly-virus contaminated homogenate in a model assay. This protocol can detect the four viruses in one assay and yield faster results for a vast number of samples, and greatly save the labor for seed potato quarantine and field surveys.

Keywords: Bulked test; High-throughput detection; Melt curve analysis; Potato viruses.

High-elevation regions in the United States lack detailed atmospheric wet-deposition data. The National Atmospheric Deposition Program/National Trends Network (NADP/NTN) measures and reports precipitation amounts and chemical constituent concentration and deposition data for the United States on annual isopleth maps using inverse distance weighted (IDW) interpolation methods. This interpolation for unsampled areas does not account for topographic influences. Therefore, NADP/NTN isopleth maps lack detail and potentially underestimate wet deposition in high-elevation regions. The NADP/NTN wet-deposition maps may be improved using precipitation grids generated by other networks. The Parameter-elevation Regressions on Independent Slopes Model (PRISM) produces digital grids of precipitation estimates from many precipitation-monitoring networks and incorporates influences of topographical and geographical features. Because NADP/NTN ion concentrations do not vary with elevation as much as precipitation depths, PRISM is used with unadjusted NADP/NTN data in this paper to calculate ion wet deposition in complex terrain to yield more accurate and detailed isopleth deposition maps in complex terrain. PRISM precipitation estimates generally exceed NADP/NTN precipitation estimates for coastal and mountainous regions in the western United States. NADP/NTN precipitation estimates generally exceed PRISM precipitation estimates for leeward mountainous regions in Washington, Oregon, and Nevada, where abrupt changes in precipitation depths induced by topography are not depicted by IDW interpolation. PRISM-based deposition estimates for nitrate can exceed NADP/NTN estimates by more than 100% for mountainous regions in the western United States.