[Value of magnetic resonance imaging texture analysis in evaluating renal allograft injury].

Objective: To explore the value of magnetic resonance imaging (MRI) texture analysis in evaluating renal allograft injury. Methods: A retrospective review was performed on sixty-six patients who underwent allograft renal transplantation (42 males, 24 females; age range, 22-63 years; mean age, (40±10) years) between November 2013 and December 2016. All the patients were divided into three groups according to their eGFR on the day of MRI examination: normal renal allograft function (nRAF) group (n=15), mild to moderate renal allograft injury (mRAI) group (n=18), and severe renal allograft injury (sRAI) group (n=33). All the patients underwent conventional T(2) weighted image (T(2)WI), susceptibility weighted imaging (SWI), and blood-oxygen level dependent (BOLD) MRI examination. MRI texture features of renal allograft were extracted. The texture features based on T(2)WI, SWI, and BOLD with absolute correlation coefficient of eGFR greater than or equal to 0.3 (P<0.05)and also with the highest Z value for Boruta algorithmwere selected. The diagnostic performance of the selected texture features in differentiating the three groups was assessed by receiver operating characteristic (ROC) curve analysis. Results: T(2)WI_Perc.50%, SWI_Perc.01%, BOLD_S(4,4)Contrast, and BOLD_S(5,5)Correlat with absolute correlation coefficient of eGFR greater than or equal to 0.3 (P<0.05) and also with the highest Z value for Boruta algorithm were selected. The AUC for T(2)WI_Perc.50%, SWI_Perc.01%, and BOLD_S(5,5)Correlat in differentiating the nRAF group with the mRAI group was 0.785, 0.720, and 0.700. The AUC for T(2)WI_Perc.50%, SWI_Perc.01%, BOLD_S(4,4)Contrast, and BOLD_S(5,5)Correlat in differentiating the nRAF group with the sRAI group was 0.687, 0.733, 0.784, and 0.737.The AUC for BOLD_S(4,4) Contrast in differentiating the mRAI group with the sRAI group was 0.667. Conclusion: MRI texture analysis can provide valuable information for evaluating renal allograft injury.

Mitochondrial fission is necessary for mitophagy, development and virulence of the insect pathogenic fungus Beauveria bassiana.

AIMS: Mitochondrial fission is an essential dynamics that maintains mitochondrial morphology and function. This study seeks to determine the roles of mitochondrial fission in the filamentous entomopathogenic fungus Beauveria bassiana. MATERIAL AND METHODS: Three fission-related genes (BbFis1, BbMdv1 and BbDnm1) were functionally characterized via protein intracellular localization and construction of gene disruption mutants. RESULTS: Mitochondrial localization was only observed for BbFis1 which interacts with BbMdv1, but BbMdv1 did not have interaction with BbDnm1. Single disruption mutant of three genes generated the elongated and enlarged mitochondria which could not be eliminated via the mitophagy. Three mutant strains displayed the reduced ATP synthesis and vegetative growth compared with the wild type. Three genes were involved in the early stage of conidiation and unnecessary for the late stage. However, all three genes significantly contribute to blastospore development under submerged condition, and the loss of BbMdv1 had the greatest effects compared with the losses of BbFis1 or BbDnm1. Finally, disruption of three genes significantly attenuated fungal virulence, but their mutations had different influences. CONCLUSIONS: In addition to their consistent roles in mitochondrial division and mitophagy, three fission-related genes perform divergent roles in the development and virulence of the entomopathogenic fungus B. bassiana. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that mitochondrial fission is associated with lifecycle of B. bassiana. These findings provide information for the manipulation of fungal physiology and facilitate the application of entomopathogenic fungi.

[The value of DCE-MRI in predicting IDH gene mutation of high-grade gliomas].

Objective: To investigate the value of quantitative and semiquantitative parameters of DCE-MRI in predicting IDH gene mutation of high-grade gliomas before the operation. Methods: Twenty-six individuals with surgically and pathologically proved WHO Ⅲ-Ⅳ gliomas collected from April 2016 to June 2019 in First People's Hospital of Changzhou, were divided into two groups, IDH mutation group (7 cases, 27-67 years, 3 males and 4 females,) and IDH gene wild group (19 cases, 42-75 years, 12 males and 7 females) according to the results of molecular pathology. All individuals underwent conventional plain (T(1)WI, T(2)WI), enhanced MR scanning (T(1)WI) and dynamic contrast enhancement (DCE). Four quantitative parameters:volume transfer constant (K(trans)), ratio constant of back flux (Kep), extravascular extracellular space fractional volume (Ve), and blood plasma fractional volume (Vp), and four semiquantitative parameters: time to peak (TTP), maximum concentration (MAX Conc), initial area under the gadolinium concentration-time curve (IAUC) and maximum slope of decrease (MAX Slope) were measured. The independent samples t test (normal distribution and homogeneity of variance) or Mann-Whitney rank sum test (abnormal distribution or heterogeneity of variance) were used to compare the differences of quantitative and semiquantitative parameters between IDH gene mutation group and IDH gene wild type group. Receiver operating characteristic (ROC) curve was used to evaluate the efficiency of quantitative and semiquantitative parameters in predicting IDH gene mutation of high-grade gliomas. Results: The value of K(trans),TTP in IDH mutated group were 0.096 (0.080,0.135)/min and (3.95±0.34) s, respectively. The value of K(trans), TTP in IDH wild type group were 0.168 (0.132, 0.337)/min and (2.58±1.15) s, respectively. The value of K(trans) in IDH mutated group was significantly less than the value of K(trans) in IDH gene wild type group (Z value was -2.168, P value was 0.030). The value of K(trans) in IDH mutated group was significantly greater than the value of K(trans) in IDH gene wild type (Z value was -2.630, P value was 0.007). The area under the ROC curve (AUC) of K(trans) and TTP in predicting IDH gene mutation of high-grade gliomas was 0.782 and 0.842, respectively. The specificity of K(trans) was higher (73.7%), The sensitivity of TTP was the higher (100.0%). Combined K(trans)and TTP were the best for predicting IDH gene mutation of high-grade gliomas, AUC was 0.865. Conclusion: Quantitative and semiquantitative parameters of DCE-MRI can help to predict IDH gene mutation of high-grade gliomas before the operation.

[Feasibility study of blood oxygen level-dependent magnetic resonance imaging in evaluating the response of metastatic lymph nodes of rabbit VX2 tumor to radiotherapy].

Objective: To investigate the feasibility of blood oxygen level-dependent magnetic resonance imaging (MRI) in evaluating the response of metastatic lymph nodes of rabbit VX2 tumor to radiotherapy. Methods: Twenty-eight healthy New Zealand white rabbits which were provided by the Laboratory Animal Center of Soochow University, male or female, 2 to 3 months, weighing 2 to 3 kg, were used to establish the animal model of VX2 tumor popliteal fossa metastatic lymph node, and then were divided into either the radiotherapy group (n=16) or the control group (n=12). The radiotherapy group received a 20 Gy radiotherapy per rabbit, the control group received sham radiotherapy. All rabbits underwent MRI scan on four time points, including before (0 day), 3rd, 7th and 14th days after radiotherapy. The two parameters of size and R(2*) value (s(-1)) of lymph node were measured. At each time point,two rabbits in each group were sacrificed randomly to resect lymph nodes for pathological examination, and two parameters of microvessel density (MVD, strip/HP) and apoptosis index (AI, %) were analyzed. The parameters among the four time points in each group or between the two groups were compared. The correlation of lymph node size and R(2*) value with MVD or AI was analyzed, respectively. Results: A significant size difference was neither between the two groups or among the each time points in each group (P>0.05). The R(2*) of lymph node in the radiotherapy group was (29.6±1.7),(36.8±2.6),(44.8±5.8) and (57.7±6.2) s(-1) at the time points of 0, 3, 7 and 14 days, respectively, showing a gradual increase trend; MVD was (52.3±2.5),(41.0±3.6),(34.0±3.6) and (22.7±2.5) strip/HP respectively, showing a decreasing trend; AI was 12.8%±0.5%,14.9%±0.6%,20.6%±0.5% and 27.5%±0.7% respectively, showing a gradual increase trend (all P<0.05). In the control group, both R(2*) value and AI among the four time points did not change statistically (all P>0.05), but MVD showed a gradual increase trend,(50.0±3.0),(53.0±1.7),(60.3±2.5) and (70.0±2.0) strip/HP, respectively, P<0.05. There were significant differences in R(2*) and MVD at 3, 7 and 14 days, in AI at 7 and 14 days between the two groups (all P<0.05). There was a linear correlation of R(2*) value, but not of size, with MVD and AI (r=-0.87 and 0.94, respectively). Conclusion: Blood oxygen level-dependent MRI can indirectly reflect the hypoxic status of metastatic lymph nodes after radiotherapy, and has potential value in evaluating the response of metastatic lymph nodes to radiotherapy.

Comparison of outcomes between off-pump versus on-pump coronary artery bypass surgery in elderly patients: a meta-analysis.

The aim of this study was to analyze if off-pump coronary artery bypass surgery (CABG) is associated with better treatment outcomes in elderly patients (>70 years of age) than on-pump CABG, using meta-analysis. Medline, PubMed, Cochrane and Google Scholar databases were searched until September 13, 2016. Sensitivity and quality assessment were performed. Twenty-two studies, three randomized control trials (RCTs) and 20 non-RCTs were included with 24,127 patients. The risk of death associated with on-pump or off-pump CABG in the RCTs were similar (pooled OR=0.945, 95%CI=0.652 to 1.371, P=0.766). However, in the non-RCTs, mortality risk was lower in patients treated with off-pump CABG than on-pump CABG (pooled OR=0.631, 95%CI=0.587 to 0.944, P=0.003). No differences were observed between the two treatment groups in terms of the occurrence of 30-day post-operative stroke or myocardial infarction (P≥0.147). In the non-RCTs, off-pump CABG treatment was associated with a shorter length of hospital stay (pooled standardized difference in means=-0.401, 95%CI=-0.621 to -0.181, P≤0.001). The meta-analysis with pooled data from non-RCTs, but not RCTs, found that mortality was lower with off-pump compared with on-pump CABG, and suggested that there may be some benefit of off-pump CABG compared with on-pump CABG in the risk of mortality and length of hospital stay.

Application of 3D printing technology on the treatment of complex proximal humeral fractures (Neer3-part and 4-part) in old people.

PURPOSE: This study was conducted to investigate the feasibility and clinical potential of using the 3D printing technology (3DPT) versus typical strategy (thin-layer CT scan) for the treatment of complicated proximal humeral fractures (PHFs) in old people. METHODS: Sixty-six old patients age ranging from 61 to 76 years with persistent complicated PHFs were randomly assigned to two groups as per the controlled randomization table (34 cases in the test group and 32 cases in the control group). In the test group, 3DPT was applied to build the 3D facture model of a patient, according to the data acquired from the thin-layer CT scan and subsequently processed with Mimics software. This helped to confirm the diagnosis, design the individual operation plan, simulate the surgical procedures and perform the surgery as plan. In the control group, only thin-layer CT scan was applied for the design of the operation plan prior to the surgery. Here, parameters including surgery duration, blood loss volume during surgery, the number of fluoroscopy, time to union were statistically analyzed for two groups after the operation. The screw lengths designed before the surgery and measured during the surgery were compared. RESULTS: The 3D PHF model generated using 3DPT was able to provide the visual display and omni-directional observation of the direction and severity of the fracture dislocation, which facilitated preoperative diagnosis, operation planning and design, data measurement, preselection of internal fixator and surgical outcome simulation. According to the follow-up ranging from 12∼28 months for the 66 patients, the results showed no significant difference in time to union between the two groups (P>0.05). Apart from that, less surgery duration, less blood loss during surgery, less number of fluoroscopy can be observed compared with the control group (P<0.05). CONCLUSIONS: In this study, 3DPT showed great clinical feasibility of the treatment of complicated PHFs. The 3D-print PHF model had the ability to clearly display the fracture and thus was useful to determine the fracture classification and the magnitude of fracture injury. It benefited surgeons to gain a better understanding of complicated PHFs, design a most suitable operation plan prior to surgery and facilitate the doctor-patient communication. This therefore enabled the reduction of intraoperative injury and the optimization of surgical outcomes.

SAG-UPS attenuates proapoptotic SARM and Noxa to confer survival advantage to early hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a deadly cancer because of its commonly late diagnosis and limited treatment options. SAG (sensitive to apoptosis gene)-dependent UPS (ubiquitin-proteasome system) is a key switch between immune-mediated apoptosis and overactivation-mediated protumorigenesis, prompting us to hypothesize that SAG-UPS modulates chronic inflammation-induced tumorigenesis. Here, we investigated the molecular mechanism by which SAG-UPS regulates death/survival of liver cancer cells. By retrospective studies, we found reciprocal expressions of anti-/proapoptotic factors: SAG/SARM and SAG/Noxa in human primary HCC tissues - the antiapoptotic SAG was significantly upregulated whereas the proapoptotic SARM and Noxa were markedly downregulated, suggesting their involvement in hepatocarcinogenesis. Upregulated SAG-UPS effectively manipulates the levels of high-molecular-weight ubiquitinated SARM and Noxa in carcinoma tissues compared with corresponding normal tissues. SAG-overexpressing HCC cell lines display reduced SARM and Noxa (but not Bcl-2, Bax and Bcl-xL), suggesting that SARM and Noxa are specific substrates of SAG-dependent ubiquitination. SARM overexpression activated caspase-3 and caspase-9, reducing cell viability. SAG knockdown significantly elevated apoptosis with increased cytosolic cytochrome c, confirming SAG-mediated antiapoptosis in HCC. SAG overexpression stimulated protumorigenic cytokines, IL-1ß, IL-6 and TNF, but not antitumorigenic IL-12p40 and anti-inflammatory IL-10. This is consistent with higher proinflammatory cytokines (IL-1ß, IL-6 and TNF) in hepatoma compared with healthy tissues. Altogether, early stage-upregulated SAG-UPS exacerbates hepatocarcinogenesis progression, through: (1) ubiquitination-mediated degradation of proapoptotic SARM and Noxa; and (2) production of protumorigenic cytokines that induce a protumorigenic microenvironment, conferring survival advantage to HCC cells. Thus, we propose SAG-UPS to be an early diagnostic marker for HCC, and a potential target for therapeutics development.

Ubiquitination by SAG regulates macrophage survival/death and immune response during infection.

The checkpoint between the life and death of macrophages is crucial for the host's frontline immune defense during acute phase infection. However, the mechanism as to how the immune cell equilibrates between apoptosis and immune response is unclear. Using in vitro and ex vivo approaches, we showed that macrophage survival is synchronized by SAG (sensitive to apoptosis gene), which is a key member of the ubiquitin-proteasome system (UPS). When challenged by pathogen-associated molecular patterns (PAMPs), we observed a reciprocal expression profile of pro- and antiapoptotic factors in macrophages. However, SAG knockdown disrupted this balance. Further analysis revealed that ubiquitination of Bax and SARM (sterile α- and HEAT/armadillo-motif-containing protein) by SAG-UPS confers survival advantage to infected macrophages. SAG knockdown caused the accumulation of proapoptotic Bax and SARM, imbalance of Bcl-2/Bax in the mitochondria, induction of cytosolic cytochrome c and activation of caspase-9 and -3, all of which led to disequilibrium between life and death of macrophages. In contrast, SAG-overexpressing macrophages challenged with PAMPs exhibited upregulation of protumorigenic cytokines (IL-1ß, IL-6 and TNF-α), and downregulation of antitumorigenic cytokine (IL-12p40) and anti-inflammatory cytokine (IL-10). This suggests that SAG-dependent UPS is a key switch between immune defense and apoptosis or immune overactivation and tumorigenesis. Altogether, our results indicate that SAG-UPS facilitates a timely and appropriate level of immune response, prompting future development of potential immunomodulators of SAG-UPS.

MR imaging of cerebral extraventricular neurocytoma: a report of 9 cases.

Extraventricular neurocytoma is a rare entity, most frequently occurring in brain parenchyma outside the ventricular system. The purpose of this study was to characterize the MR imaging findings in a series of 9 patients with EVN verified by results of pathologic examination. All 9 EVNs were solitary and intracranially located. Eight lesions were well demarcated, and 3 showed intratumoral hemorrhage. The solid parts of 7 tumors were primarily isointense on T1-weighted images and heterogeneously enhanced on T1WI with contrast. Although cerebral EVNs can present a wide spectrum of appearances on MR, the imaging patterns appear to vary according to anatomic location and cellularity. Lesions in frontal or parietal lobes often present as well-demarcated large masses with cystic degeneration, hemorrhage, mild-to-moderate edema, and inhomogeneous enhancement. Moreover, the general isointensity of the solid parts of EVN on T1WI may be of some specificity.

T-cell death following immune activation is mediated by mitochondria-localized SARM.

Following acute-phase infection, activated T cells are terminated to achieve immune homeostasis, failure of which results in lymphoproliferative and autoimmune diseases. We report that sterile α- and heat armadillo-motif-containing protein (SARM), the most conserved Toll-like receptors adaptor, is proapoptotic during T-cell immune response. SARM expression is significantly reduced in natural killer (NK)/T lymphoma patients compared with healthy individuals, suggesting that decreased SARM supports NK/T-cell proliferation. T cells knocked down of SARM survived and proliferated more significantly compared with wild-type T cells following influenza infection in vivo. During activation of cytotoxic T cells, the SARM level fell before rising, correlating inversely with cell proliferation and subsequent T-cell clearance. SARM knockdown rescued T cells from both activation- and neglect-induced cell deaths. The mitochondria-localized SARM triggers intrinsic apoptosis by generating reactive oxygen species and depolarizing the mitochondrial potential. The proapoptotic function is attributable to the C-terminal sterile alpha motif and Toll/interleukin-1 receptor domains. Mechanistically, SARM mediates intrinsic apoptosis via B cell lymphoma-2 (Bcl-2) family members. SARM suppresses B cell lymphoma-extra large (Bcl-xL) and downregulates extracellular signal-regulated kinase phosphorylation, which are cell survival effectors. Overexpression of Bcl-xL and double knockout of Bcl-2 associated X protein and Bcl-2 homologous antagonist killer substantially reduced SARM-induced apoptosis. Collectively, we have shown how T-cell death following infection is mediated by SARM-induced intrinsic apoptosis, which is crucial for T-cell homeostasis.

Single-stranded DNA oligoaptamers: molecular recognition and LPS antagonism are length- and secondary structure-dependent.

In Gram-negative bacterial infection, lipopolysaccharide (LPS) readily overwhelms the host innate immune system, which could result in inflammation and sepsis in severe cases. Therefore, developing anti-LPS molecules would confer an efficient antibacterial strategy. We used SELEX (Systemic Evolution of Ligands by EXponential enrichment) to isolate single-stranded DNA (ssDNA) aptamers. By immobilizing and exposing different orientations of the LPS molecule on hydrophobic and hydrophilic surfaces, two populations of aptamers were captured from a library of 10(14-15) ssDNA oligonucleotides. Progressive SELEX enriched the aptamers towards thymidine residues. The more hydrophobic aptamers with T-rich loops showed strong molecular recognition for the lipid A moiety of LPS, binding at affinity of up to K(D) of 10(-9)M, and eliciting 95% neutralization of endotoxicity. The longer ssDNAs exhibited greater avidity for LPS and conferred more efficacious antagonism against LPS. The nucleotide composition imposes subtle influence on the aptamer folding and affinity for LPS.

A novel serine protease inhibitor acts as an immunomodulatory switch while maintaining homeostasis.

Serine protease cascades boost immune responses while maintaining homeostasis. These crucial actions are intricately regulated by cognate serine protease inhibitors. However, the mechanism underlying such a dynamic immunomodulation during acute phase infection remains obscure, particularly where the pathogen's serine protease adds a new challenge to the host. Here, we found that infection of horseshoe crab, Carcinoscorpius rotundicauda, induced reciprocal profiles of CrSPI (serine protease inhibitor) and CrFurin (serine protease) with respect to their transcription and protein activities. Using recombinant rCrSPI, we explored its inhibitory activity against various microbial proteases and found it most efficacious against a model serine protease, subtilisin A. rCrSPI inhibited subtilisin at Ki 10(-9)M with a molar ratio of 1 rCrSPI:2 subtilisin. The rCrSPI also inhibited plasma CrFurin, suppressed subtilisin-mediated activation of prophenoloxidase (PPO) and interacted with complement C3. Taken together, CrSPI acts as a key immunomodulatory 'on-off' switch in a 2-way regulation of serine protease microbial subtilisin and host serine proteases (CrFurin and CrC3), thereby controlling immune responses involving the complements and the PPO-mediated antimicrobial activities, while maintaining homeostasis.

The Sushi peptides: structural characterization and mode of action against Gram-negative bacteria.

The compositional difference in microbial and human cell membranes allows antimicrobial peptides to preferentially bind microbes. Peptides which specifically target lipopolysaccharide (LPS) and palmitoyl-oleoyl-phosphatidylglycerol (POPG) are efficient antibiotics. From the core LPS-binding region of Factor C, two 34-mer Sushi peptides, S1 and S3, were derived. S1 functions as a monomer, while S3 is active as a dimer. Both S1 and S3 display detergent-like properties in disrupting LPS aggregates, with specificity for POPG resulting from electrostatic and hydrophobic forces between the peptides and the bacterial lipids. During interaction with POPG, the S1 transitioned from a random coil to an alpha-helix, while S3 resumed a mixture of alpha-helix and beta-sheet structures. The unsaturated nature of POPG confers fluidity and enhances insertion of the peptides into the lipid bilayer, causing maximal disruption of the bacterial membrane. These parameters should be considered in designing and developing new generations of peptide antibiotics with LPS-neutralizing capability.

Atomic force microscopy study of the antimicrobial action of Sushi peptides on Gram negative bacteria.

The antibacterial effect of the endotoxin-binding Sushi peptides against Gram-negative bacteria (GNB) is investigated in this study. Similar characteristics observed for Atomic force microscopy (AFM) images of peptide-treated Escherichia coli and Pseudomonas aeruginosa suggest that the Sushi peptides (S3) evoke comparable mechanism of action against different strains of GNB. The results also indicate that the Sushi peptides appear to act in three stages: damage of the bacterial outer membrane, permeabilization of the inner membrane and disintegration of both membranes. The AFM approach has provided vivid and detailed close-up images of the GNB undergoing various stages of antimicrobial peptide actions at the nanometer scale. The AFM results support our hypothesis that the S3 peptide perturbs the GNB membrane via the "carpet-model" and thus, provide important insights into their antimicrobial mechanisms.

Ancient paddy soils from the Neolithic age in China's Yangtze River Delta.

Identifying prehistoric irrigated rice fields and characterizing the beginning of paddy soil development are important for a better understanding of human development and agricultural history. In 2003, paddy soils and irrigated rice fields buried at a depth of 100-130 cm were excavated at Chuo-dun-shan in the Yangtze River Delta, close to Suzhou, China. The fields of sizes between 1.4 and 16 m(2) were surrounded with ridges that were connected to ditches/ponds via outlets to control the water level within the fields. Many carbonized and partly carbonized rice grains with an age of 3,903 B.C. (measured (14)C age 5,129+/-45 a BP) were recovered. The surface layers of these buried paddy fields showed a high content of soil organic matter and a considerable high density of rice opals. The latter were identified to derive from Oryza spp. Solid-state (13)C nuclear magnetic resonance spectroscopy revealed aromatic carbon (C) as the predominant organic C form in the fossil surface layer. This is expected, if the major source represents burnt rice and straw. In summary, our data are in agreement with new evidences indicating that in China, paddy soils and irrigated rice cultivation were initiated and developed more than 6,000 years ago.

The specificity of Sushi peptides for endotoxin and anionic phospholipids: potential application of POPG as an adjuvant for anti-LPS strategies.

Sushi peptides [S1 (Sushi 1 peptide) and S3] are derived from the LPS (lipopolysaccharide; also known as endotoxin)-binding domains of an LPS-sensitive serine protease, Factor C, from the horseshoe crab (Carcinoscorpius rotundicauda). S1 and S3 interact at high affinity with LPS. The intermolecular disulphide bonding in the S3 dimer is indispensable for its LPS binding, disruption and consequent neutralization. Simultaneously, the specific interaction between the Sushi peptides and bacterial membrane phospholipids further explains the selective propensity of these peptides for the gram-negative bacteria. Our findings yield insights into a complex molecular paradigm in which the juxtaposition of LPS molecules and the anionic phospholipid POPG (1-palmitoyl-2-oleoyl phosphatidylglycerol) on the bacterial outer membrane confers such interfacial properties which create the optimal environment for the interaction between the peptides and bacterial membrane lipids.

Spatial and temporal coordination of expression of immune response genes during Pseudomonas infection of horseshoe crab, Carcinoscorpius rotundicauda.

Knowledge on how genes are turned on/off during infection and immunity is lacking. Here, we report the co-regulation of diverse clusters of functionally related immune response genes in a horseshoe crab, Carcinoscorpius rotundicauda. Expressed sequence tag (EST) clusters for frontline immune defense, cell signalling, apoptosis and stress response genes were expressed or repressed spatio-temporally during the acute phase of Pseudomonas infection. An infection time course monitored by virtual Northern evaluation indicates upregulation of genes in blood cells (amebocytes) at 3-h postinfection, whereas most of the hepatopancreas genes remained down regulated over 72 h of infection. Thus, the two tissues orchestrate a coordinated and timely response to infection. The hepatopancreas probably immuno-modulates the expression of other genes and serves as a reservoir for later response, if/when chronic infection ensues. On the other hand, being the first to encounter pathogens, we reasoned that amebocytes would respond acutely to infection. Besides acute transactivation of the immune genes, the amebocytes maintained morphological integrity, indicating their ability to synthesise and store/secrete the immune proteins and effectors to sustain the frontline innate immune defense, while simultaneously elicit complement-mediated phagocytosis of the invading pathogen. Our results show that the immune response against Pseudomonas infection is spatially and temporally coordinated.

Single-cell protein diet of a novel recombinant vitellogenin yeast enhances growth and survival of first-feeding tilapia (Oreochromis mossambicus) larvae.

Yeast single-cell protein (SCP) is a high-nutrient feed substitute. This study evaluates the dual applications of a novel recombinant Pichia pastoris SMD1168H (SMD) yeast, expressing a tilapia vitellogenin protein (rVtg), as an SCP diet for Artemia and the first-feeding fish larvae. Instar II Artemia fed rVtg, rVtg precultured in 5% fish oil (rVtg-FO), Saccharomyces cerevisiae (SC), or native SMD had greater lipid contents (P < 0.05) than the freshly hatched. Lipid deposition in the Artemia fed rVtg or rVtg-FO was greater (P < 0.05) than in those fed SMD or SC. Diet-induced accumulation of low levels of docosahexaenoic acid [22:6(n-3)] was detected only in Artemia fed the rVtg-based diets. Tilapia (Oreochromis mossambicus) larvae were fed solely yeast diets singly or in combination (d 3-22), or a staggered regimen of yeast (d 3-12) followed by unenriched or yeast-enriched Artemia (d 13-22). The larvae fed rVtg for 22 d increased in length and weight (P < 0.05), whereas those fed SC or SMD suffered growth suppression and high mortality. Such adverse consequences were ameliorated when 50% of SC was substituted with rVtg. The larvae prefed rVtg followed by a dietary switch to Artemia preenriched for 48 h with rVtg or rVtg-FO were greatest in length, had the highest weight gain, and lived the longest. Besides delivering rVtg protein, essential fatty acids and amino acids, rVtg may have probiotic effects in enhancing larval survival. This study suggests the feasibility of using the rVtg yeast as an Artemia booster and an SCP first feed for larvae.

De novo design of potent antimicrobial peptides.

Lipopolysaccharide (LPS), shed by gram-negative bacteria during infection and antimicrobial therapy, may lead to lethal endotoxic shock syndrome. A rational design strategy based on the presumed mechanism of antibacterial effect was adopted to design cationic antimicrobial peptides capable of binding to LPS through tandemly repeated sequences of alternating cationic and nonpolar residues. The peptides were designed to achieve enhanced antimicrobial potency due to initial bacterial membrane binding with a reduced risk of endotoxic shock. The peptides designed displayed binding affinities to LPS and lipid A (LA) in the low micromolar range and by molecular modeling were predicted to form amphipathic beta-hairpin-like structures when they bind to LPS or LA. They also exhibited strong effects against gram-negative bacteria, with MICs in the nanomolar range, and low cytotoxic and hemolytic activities at concentrations significantly exceeding their MICs. Quantitative structure-activity relationship (QSAR) analysis of peptide sequences and their antimicrobial, cytotoxic, and hemolytic activities revealed that site-directed substitutions of residues in the hydrophobic face of the amphipathic peptides with less lipophilic residues selectively decrease the hemolytic effect without significantly affecting the antimicrobial or cytotoxic activity. On the other hand, the antimicrobial effect can be enhanced by substitutions in the polar face with more polar residues, which increase the amphipathicity of the peptide. On the basis of the QSARs, new analogs that have strong antimicrobial effects but that lack hemolytic activity can be proposed. The findings highlight the importance of peptide amphipathicity and allow a rational method that can be used to dissociate the antimicrobial and hemolytic effects of cationic peptides, which have potent antimicrobial properties, to be proposed.