Acute rejection is associated with antibodies to non-Gal antigens in baboons using Gal-knockout pig kidneys

We transplanted kidneys from alpha1,3-galactosyltransferase knockout (GalT-KO) pigs into six baboons using two different immunosuppressive regimens, but most of the baboons died from severe acute humoral xenograft rejection. Circulating induced antibodies to non-Gal antigens were markedly elevated at rejection, which mediated strong complement-dependent cytotoxicity against GalT-KO porcine target cells. These data suggest that antibodies to non-Gal antigens will present an additional barrier to transplantation of organs from GalT-KO pigs to humans.     

Development and validation of EST-derived SSR markers and diversity analysis in cluster bean (<Emphasis Type="Italic">Cyamopsis tetragonoloba</Emphasis>)

Cluster bean/guar (Cyamopsis tetragonoloba), has an important place in industry because of its seeds, which contain galactomannan (guar gum) rich endosperm. Guar gum, an important ingredient of many products, is purely an export oriented commodity. Development of molecular markers for this crop is essential to accelerate breeding for guar gum content in seeds. A total of 100 novel primers pairs were developed from 16,476 expressed sequence tags (ESTs) sequence of cluster bean. A total of 50 primers pairs with function annotation of gum synthesis were selected and validated on a panel of 32 genotypes. Among the 50 primers 39 primers were amplified with a total of 45 loci. The polymorphic information content (PIC) ranged from 0.00 to 0.42 with an average of 0.13. With low polymorphic simple sequence repeats (SSRs) and narrow genetic base, most of the genotypes scattered into three clusters regardless of their geographical origin. Present study showed the existence of very low genetic diversity in cluster bean. The results indicated that there is need to explore SSR markers from whole genome or alternative marker systems like SNP (single nucleotide polymorphism) markers, for effective implication of markers in cluster bean breeding.     

New phenolic inhibitors of the peroxidse-catalysed oxidation of indole-3-acetic acid

Previously we reported two metabolites of the insecticide carbofuran as persistent inhibitors of the peroxidase-catalysed oxidtion ofindole-3-acetic acid. In searching for more active inhibitors of this type, we have found that 5-hydroxy-2,2-dimethylchromene (β-tubanol), 2′,6′-dihydroxycetophenone oxime, 5-hydroxy-2,2-dimethylchroman, 2′,6′-dihydroxyacetophenone and 2,6-dihydroxybenzoic acid methyl ester were more active than the carbofuran metabolite 7-hydroxy-2,2-dimethyl-3-oxo-2,3-dihydrobenzofuran. Resorcinol, 5-hydroxy-2,2-dimethylchroman-4-one, 3-hydroxy-5-methoxy-2,2-dimethylchroman-4-one and 5-hydroxy-2-methylchrom-4-one were also inhibitory but with less activity. The new inhibitors differed from the well-known phenolic inhibitors such as caffeic acid in inhibition kinetics as demonstrated by the rate of disappearance of indole-3-acetic acid, the rate of formation of the oxidation products, and the transient spectral change in the enzyme.     

Biotreatment of nitrate-rich industrial effluent by suspended bacterial growth

Of the 29 potentially denitrifying organisms isolated from a denitrifying reactor (DNR) of a fertilizer company, two isolates; I-4 and I-5 were recognized as denitrifiers. Under aerobic conditions, with fusel oil as the carbon source, the organisms decreased nitrate from 1200 mg l^–1 to 100 mg l^–1 in 48 h. Optimal growth conditions for biological removal of nitrate were established in batch culture. The system was scaled up to 4-L and 50-L bioreactors under continuous culture conditions. Up to 95–100% nitrate removal was achieved in the 50-L bioreactor at a COD:NO₃–N ratio of 3.45 with a retention time of 48 h. The isolates showed 1.5 fold higher denitrifying activity than reported previously.     

I-Ag7-mediated antigen presentation by B lymphocytes is critical in overcoming a checkpoint in T cell tolerance to islet beta cells of nonobese diabetic mice.

B cell-deficient nonobese diabetic (NOD) mice are protected from the development of spontaneous autoimmune diabetes, suggesting a requisite role for Ag presentation by B lymphocytes for the activation of a diabetogenic T cell repertoire. This study specifically examines the importance of B cell-mediated MHC class II Ag presentation as a regulator of peripheral T cell tolerance to islet beta cells. We describe the construction of NOD mice with an I-Ag7 deficiency confined to the B cell compartment. Analysis of these mice, termed NOD BCIID, revealed the presence of functionally competent non-B cell APCs (macrophages/dendritic cells) with normal I-Ag7 expression and capable of activating Ag-reactive T cells. In addition, the secondary lymphoid organs of these mice harbored phenotypically normal CD4+ and CD8+ T cell compartments. Interestingly, whereas control NOD mice harboring I-Ag7-sufficient B cells developed diabetes spontaneously, NOD BCIID mice were resistant to the development of autoimmune diabetes. Despite their diabetes resistance, histologic examination of pancreata from NOD BCIID mice revealed foci of noninvasive peri-insulitis that could be intentionally converted into a destructive process upon treatment with cyclophosphamide. We conclude that I-Ag7-mediated Ag presentation by B cells serves to overcome a checkpoint in T cell tolerance to islet beta cells after their initial targeting has occurred. Overall, this work indicates that the full expression of the autoimmune potential of anti-islet T cells in NOD mice is intimately regulated by B cell-mediated MHC class II Ag presentation.     

Biochemical changes in the corpus luteum of intrauterine device fitted goats

Parous goats were sham operated (6 goats), bilaterally fitted with IUDs (7 goats), or unilaterally fitted with an IUD isilateral (6 goats) or contralateral (5 goats) to the corpus luteum (CL). Autopsy was on Day 3 or Day 5 of the second or third estrus. The cycle length of IUD-fitted goats was shorter than in controls (6.2 days vs. 16.7 days, p less than .05). Mean CL weight in the contralateral group, 164.8 mg, exceeded mean weight of the ipsilateral group, 92.4 mg (P less than .01). The concentration of total cholesterol was higher in the ipsilateral group than in the contralateral group (3.16 vs. 2.12 mg/gm CL, p less than .01). The protein concentration in CL from the control, unilateral and bilateral groups was 14.4, 12.2, and 23.9 mg, respectively, p less than .05. The content and concentration of progesterone, and total and free cholesterol was higher in sham operated controls than in IUD-fitted goats (not significant). Protein, RNA, and DNA increased considerably in IUD-fitted goats (not significant), indicating a derangement of a luteotrophic rather than luteomorphic mechanism in CL from IUD-fitted goats.     

Capturing the songs of mice with an improved detection and classification method for ultrasonic vocalizations (BootSnap)

House mice communicate through ultrasonic vocalizations (USVs), which are above the range of human hearing (>20 kHz), and several automated methods have been developed for USV detection and classification. Here we evaluate their advantages and disadvantages in a full, systematic comparison, while also presenting a new approach. This study aims to 1) determine the most efficient USV detection tool among the existing methods, and 2) develop a classification model that is more generalizable than existing methods. In both cases, we aim to minimize the user intervention required for processing new data. We compared the performance of four detection methods in an out-of-the-box approach, pretrained DeepSqueak detector, MUPET, USVSEG, and the Automatic Mouse Ultrasound Detector (A-MUD). We also compared these methods to human visual or ‘manual’ classification (ground truth) after assessing its reliability. A-MUD and USVSEG outperformed the other methods in terms of true positive rates using default and adjusted settings, respectively, and A-MUD outperformed USVSEG when false detection rates were also considered. For automating the classification of USVs, we developed BootSnap for supervised classification, which combines bootstrapping on Gammatone Spectrograms and Convolutional Neural Networks algorithms with Snapshot ensemble learning. It successfully classified calls into 12 types, including a new class of false positives that is useful for detection refinement. BootSnap outperformed the pretrained and retrained state-of-the-art tool, and thus it is more generalizable. BootSnap is freely available for scientific use.     

The development, characterization, and demonstration of a novel strategy for purification of recombinant proteins expressed in plants

Plants have attracted increasing attention as an expression platform for the production of pharmaceutical proteins due to its unlimited scalability and low cost potential. However, compared to other expression systems, plants accumulate relatively low levels of foreign proteins, thus necessitating the development of efficient systems for purification of foreign proteins from plant tissues. We have developed a novel strategy for purification of recombinant proteins expressed in plants, based on genetic fusion to soybean agglutinin (SBA), a homotetrameric lectin that binds to N-acetyl-D-galactosamine. Previously it was shown that high purity SBA could be recovered from soybean with an efficiency of greater than 90% following one-step purification using N-acetyl-D-galactosamine-agar columns. We constructed an SBA fusion protein containing the reporter green fluorescent protein (GFP) and transiently expressed it in N. benthamiana plants. We achieved over 2.5% of TSP accumulation in leaves of N. benthamiana. Confocal microscopic analysis demonstrated in vivo activity of the fused GFP partner. Importantly, high purity rSBA-GFP was recovered from crude leaf extract with ~90% yield via one-step purification on N-acetyl-D-galactosamine-agar columns, and the purified fusion protein was able to induce the agglutination of rabbit red blood cells. Combined with this, tetrameric assembly of the fusion protein was demonstrated via western blotting. In addition, rSBA-GFP retained its GFP signal on agglutinated red blood cells, demonstrating the feasibility of using rSBA-GFP for discrimination of cells that bear the ligand glycan on their surface. This work validates SBA as an effective affinity tag for simple and rapid purification of genetically fused proteins.     

LFA-1 fine-tuning by cathepsin X

The adhesion molecule lymphocyte function-associated antigen (LFA)-1 plays a key role in immune surveillance and response. Its conformation is spatially and temporally regulated, enabling adhesion and deadhesion during T-cell migration. LFA-1 adhesion to its major ligand intercellular adhesion molecule 1 is controlled by adaptor proteins which bind the cytoplasmic tail of the β (2) subunit. Cathepsin X, a cysteine carboxypeptidase, promotes T-cell migration and morphological changes by cleaving the β (2) cytoplasmic tail of LFA-1. In this way, it modulates the affinity of LFA-1 for structural adaptors talin-1 and α-actinin-1 and enables the stepwise transition between intermediate and high-affinity conformations of LFA-1, an event that is necessary for effective T-cell function. Cathepsin X regulation that would allow precise modulation of LFA-1 affinity has a great potential for anti-LFA-1 therapy.